Soft Palate Dimensions and Nasopharyngeal Depth (Need’s Ratio) In Different Sagittal and Vertical Skeletal Patterns: A Lateral Cephalometric Study-JuniperPublishers

Journal of Dentistry & Oral Health-Juniper Publishers

Abstract

Objectives: Are to compare the soft palate length (SPL), width and nasopharyngeal depth (PD) in different sagittal and vertical skeletal patterns and to see the influence of different skeletal malocclusions on need’s ratio (PD/SPL).

Material & Methods: Lateral cephalograms of 372 patients were equally divided into sagittal (class I, II, III) and vertical skeletal patterns (normodivergent, hypodivergent, hyperdivergent) by measuring ANB and SN-MP angles, respectively. SPL, velar width (VW) and PD were recorded using Rogan Delft View Pro-X software. Kruskal Wallis test was used to compare SPL, VW, PD and need’s ratio between sagittal and vertical skeletal groups. Intergroup comparisons were performed using Mann-Whitney U-test. Level of significance was kept at p≤0.05.

Results: Statistically significant differences were found for VW (p=0.008) and need’s ratio (p=0.035) amongst sagittal groups. Amongst vertical groups, significant differences were found for SPL (p< 0.001), VW (p=0.021) and needs ratio (p=0.020). On intergroup comparison, SPL (p=0.031), VW (p=0.011) and Need’s ratio (p=0.013) were significantly different between skeletal class I and III and VW (p = 0.005) between skeletal class II and III malocclusions. The SPL was significantly different between normodivergent and hypodivergent (p=0.004) and normodivergent and hyperdivergent groups (p< 0.001). The VW was significantly different between hyperdivergent and hypodivergent groups (p=0.005) and needs ratio between normodivergent and hyperdivergent groups (p=0.001). Gender difference was significant for SPL which was larger in males as compared to females in skeletal class III malocclusion (p=0.001).

Conclusion: Soft palate length, width and need’s ratio may vary with the underlying skeletal malocclusion

Keywords: Need’s ratio; Soft palate; Lateral cephalogram

Introduction

The roof of the mouth anatomically separates the nasal cavity from the oral cavity and structurally is composed of anterior bony component i.e. hard palate and posterior fibro-muscular component i.e. soft palate (also known as velum) [1,2]. The contribution of the soft palate towards velopharyngeal closure is related to normal oral functions of sucking, deglutition, and articulation [3]. The velopharynx is a roughly rectangular space which is bordered anteriorly by velum (soft palate), posteriorly by posterior pharyngeal wall, and laterally by right and left lateral pharyngeal walls [4]. The contractions of these structures assist in closure of velopharyngeal port during the acts of eating, swallowing and speaking whereas; their relaxation opens the port for breathing and in producing specific nasalized articulations. A close coordination of the soft palate with the posterior pharyngeal wall is important during pronouncing most of the vowels and consonants [3] The failure of velopharyngeal sphincter mechanism to perform these aforementioned functions results in velopharyngeal dysfunction [4-6].

The relationship between soft palate length (SPL) and nasopharyngeal depth (PD) can be used to determine the velopharyngeal function and is called the Need’s ratio (PD/SPL). According to Subtelny [7], the Need’s ratio should be in a range of 0.6-0.7 in normal subjects. Likewise, Simpson & Colton [8] and Hoopes et al. [9] found the normal ratio to be 0.75 to 0.8. Any increase greater than 80% demonstrated a risk for developing velopharyngeal insufficiency [10]. This ratio is of paramount interest in detecting speech related problems and can be influenced by dentofacial orthopedics, adenoidectomy, uvulopalatopharyngoplasty and maxillary advancement surgeries [11].

Soft palate dysfunctions are most commonly observed in cleft palate patients who have short or otherwise abnormal velum, sub mucous cleft palate, excessively large tonsils or adenoids, webbing of the posterior tonsillar pillars, obstructive sleep apnea or skeletal craniofacial malocclusions [2,12]. There have been studies in the literature that have evaluated the soft palate and airway dimensions in different sagittal and vertical skeletal malocclusions [13-15], but little work has been done to see the relationship of soft palate length with the nasopharyngeal depth (need’s ratio) in these malocclusions [15].

Hence, the objective of this study was to compare the soft palate length, width and nasopharyngeal depth in different sagittal (skeletal class I, II and III malocclusions) and vertical (normodivergent, hypodivergent and hypedivergent) skeletal patterns and to see if the need’s ratio is affected by changes in the underlying skeletal patterns.

Since a number of adult patients who seek orthodontic treatment may require orthognathic surgeries as well as multiple cleft patients with hypernasal speech may undergo Le-Fort I maxillary advancement, the pre-surgical assessment of the need’s ratio is important. This will help us in planning appropriate jaw movements during orthognathic surgeries in treatment of underlying sagittal skeletal discrepancies. In addition, patients presented with moderate to severe skeletal class II malocclusion with pre-existing enlarged adenoids or tonsils, allergies, asthma or obesity may develop obstructive sleep apnea (OSA) in future [16]. Knowledge about the difference in soft palate length and width, nasopharyngeal dimensions and Need’s ratio in different skeletal malocclusions will assist in better understanding of the etiology of OSA syndrome.

Materials and Methods

Sample size was calculated by using NCSS statistical software (version 13), keeping α = 0.05, power of study (β) as 80% and by using the findings of a study conducted by Abu Allhaija & Al- Khateeb [13]. They reported the mean and SD of soft palate length (PNS-P) amongst the three skeletal malocclusions as (class I= 36.7 ± 3.7, class II= 37.2 ± 4.6, class III= 34.7 ± 4.7). A power analysis showed that we required a minimum sample of 62 subjects for one group. Since, there were three sagittal groups (skeletal class I, II and III) and three vertical groups (normodivergent, hypo divergent and hyper divergent) in this study; a total sample calculated was 372 subjects.

Ethical clearance was obtained from the institutional Ethical review Committee, prior to the data collection. The inclusion criteria of this study were pre-treatment lateral cephalograms of adult patients seeking orthodontic treatment, aged 16-35 years. A total of 372 Lateral cephalograms of the patients were equally divided into sagittal (class I, II and III) and vertical skeletal patterns (normodivergent, hypodivergent and hyper divergent) on the basis of ANB and SN-MP angles, respectively. For the sagittal groups, this was assessed on pre-treatment lateral cephalometric tracings by measuring ANB angle (Figure 1). The ANB angle was set at 1-4°, >5° and < 0° for skeletal class I, II and III malocclusions, respectively. The vertical malocclusion groups were categorized by measuring SN-MP angle into normodivergent (SN-MP = 33- 37°), hypo divergent (SN-MP < 32°), and hyper divergent facial patterns (SN-MP > 38°) as shown in Figure 2.

The exclusion criteria of this study were radiographs of patients with cleft palate, any systemic disease that may affect head and neck region, craniofacial syndromes, fractures of head and neck, pharyngeal pathology, nasal obstruction, enlarged tonsils or adenoids and any previous history of orthodontic treatment.

To keep a high degree of precision, all the pre-treatment lateral cephalograms of subjects were routinely taken with the sagittal plane at right angle to the path of x-ray beams, the head in an erect position, Frankfort horizontal plane being parallel to the horizontal, teeth in centric occlusion and lips closed in a relaxed position. These radiographs were recorded with rigid head fixation and a 165-cm film-to-tube distance using Orthoralix R 9200 (Gendex-KaVo, Milan, Italy).

Lateral cephalograms were viewed and analyzed digitally on Rogan Delft View Pro-X software. The soft palate length (SPL) was measured as a linear distance from the posterior nasal spine (PNS) to the tip of the uvula of the resting soft palate. The velar width (VW) was measured at the thickest section of the velum. The nasopharyngeal depth (PD) was taken as a linear measurement from the posterior nasal spine to the posterior pharyngeal wall along the palatal plane (Figure 3). The Need’s ratio was calculated for all the subjects by dividing the pharyngeal depth (PD) with soft palate length (SPL). To avoid the examiner bias, 20 lateral cephalograms were randomly selected after a month and reevaluated to assess the intra-examiner reliability.

Statistical Analysis

All the statistical analyses of data were performed using the SPSS for Windows (version 19.0, SPSS Inc. Chicago). Shapiro-Wilk test was used to explore the normality of the data which showed a non-normal distribution for most of the variables. Descriptive statistics such as mean and SD were calculated for the soft palate length, width, nasopharyngeal depth and Needs ratio (PD/SPL) and Kruskal Wallis test was used to compare these variables between sagittal and vertical skeletal patterns. Intergroup comparisons between different sagittal and vertical skeletal patterns were performed using Mann-Whitney U-test. For intersex comparison, similar statistical tests were performed. Level of significance was kept at p ≤ 0.05

Results

A total sample comprised of 372 subjects who were further divided equally into sagittal groups [class I = 62, class II = 62 and class III = 62] and vertical groups (normodivergent = 62, hypodivergent = 62, hyperdivergent = 62]. Each sagittal and vertical malocclusion group was further subdivided into 31 males and 31 females. Descriptive statistics such as mean and SD was calculated for chronological age of the subjects. The mean age of subjects in skeletal class I, II and II malocclusions were 22.4 ± 6.04, 21.2 ± 5.67 and 19.3 ± 4.13 years, respectively. In vertical malocclusion groups, the mean age of subjects were 18.8 ± 3.07, 22.5 ± 6.03 and 22.7 ± 6.93 for normodivergent, hypodivergent and hyperdivergent skeletal patterns, respectively.

Table 4 presents the intersex comparison of the variables. The velar length was significantly larger in males as compared to females in skeletal class III malocclusion (p = 0.001). The other quantitative variables did not show significant gender differences.

To determine the intra-examiner reliability for the repeated measurements, Intra class Correlation Coefficients (ICC) was applied. The coefficients obtained were above 0.9 for all the variables, confirming the reliability of the repeated measurements.

Discussion

Lateral cephalometric radiographs have been used since many years for evaluating soft palate and nasopharyngeal dimensions in patients with obstructive sleep apnea and cleft palate as well as in normal individuals. In addition, several studies have assessed the of the soft palate and superior pharyngeal space with more sophisticated and advanced radiographic techniques such as cine-computed tomography (CT), nasopharyngoscopy, videofluoroscopy and magnetic resonance imaging (MRI) [15,17,18]. However, due to their increased cost and high radiation dose, they are better reserved for patients with some known soft palate dysfunctions or velopharnygeal incompetency.

Lateral cephalometric radiograph is relatively less expensive and are more useful in patients seeking orthodontic treatment, have reduced radiation exposure and provides good visibility of the soft palate and its surrounding structures [19]. In addition, it also reveals a variety of craniofacial characteristics which are often associated with patients of obstructive sleep apnea [18]. Jhonston & Richardson [20] found that small and retrognathic skeletal structures, reduced airway and increased length and width of soft palate poses a risk for developing OSA. In our study, Type 5 (S-shaped) soft palate was longest and found in 9.1% of the total patients, the vast majority of which had skeletal class II malocclusion. The increased frequency of S-shaped soft palate (which was found to be the longest soft palate type) in skeletal class II malocclusion patients who have retrognathic jaws may further add the risk of developing OSA. The S-shaped soft palate was observed in 4.7% of the subjects in a study conducted by Verma et al. [21], 3.5 % in You et al. [12] research and in 1.5% of the cases in Guttal et al. [22] study.

The present study used Rogan Delft View Pro X software for morphological assessment of soft palate as well as its dimensions on lateral cephalogram in different skeletal malocclusions. This software enabled us to use the magnification tool for better visualization of the soft palate type as well as allowed us to adjust and optimize the contrast and gradation. The soft palate and nasopharyngeal dimensions in different skeletal malocclusions have been investigated in several previous studies [13-15]. Abu Allhaija and Al-Khateeb [13] did not find any significant differences for the soft palate length, soft palate thickness and nasophryngeal dimensions amongst skeletal Class I, II and III malocclusions. Likewise, a study conducted by Soheilifar et al [14] reported insignificant differences for soft palate dimensions and pharyngeal depth between skeletal class I and II subjects. In our study, we found statistically significant differences for the soft palate length and thickness between skeletal class I and III and soft palate width between skeletal class II and III malocclusions. The soft palate was largest in skeletal class I and widest in skeletal class III malocclusion. However, the differences in nasopharyngeal depth were insignificant.

The growth of the soft palate in length with an increasing age has been investigated by Suntelny [7]. He found a rapid increase in growth during the early years of life which levels off till the age of 5 years. However, the growth of nasopharyngeal dimensions remain continue till 13 years and then plateau till adulthood [13,23]. To rule out the influence of growth on these structures, we have included adult subjects in this study. This ensures that the soft palate length and pharyngeal depth of all the subjects had almost reached their maximum size.

For intersex comparisons, Subtelny [7] in his study found an increased length of soft palate in males as compared to females. In contrast, Abu Allhaija and Al-Khateeb [13] didn’t find any significant gender differences in soft palate and nasopharyngeal dimensions in different skeletal malocclusions. In our study, males had increased velar length in skeletal class III malocclusion group as compared to the females. In skeletal class I and class II, no significant differences were observed for any of the study variables across the gender.

An increase in this ratio above the normal range may disturb the normal velopharyngeal function which may result in problem related to speech and resonance. Short soft palates along with an increased nasopharyngeal depth are important etiological factors in developing velopharyngeal insufficiency especially in patients undergoing maxillary advancement surgeries [15]. Haapanen et al [24], found 27% of the patients with cleft palate demonstrated reduced velopharyngeal function following Le-fot I maxillary advancement procedure. Our study results showed statistically significant differences for the Need’s ratio between skeletal class I and III malocclusions.

Clinical Implications

The variation in Need’s ratio amongst different skeletal malocclusions calls attention towards thorough radiographic examination of soft palate dimensions and nasopharyngeal depth during orthodontic diagnosis and treatment planning. Any treatment plan that may affect the stability of the Need’s ratio should be avoided in order to prevent speech related problems and obstructive sleep apnea. For instance, cleft palate patients with known velopharyngeal insufficiency having severe skeletal class III malocclusion due to maxillary deficiency, a double jaw surgery approach is preferred over single jaw osteotomy. A combination of Le-Fort I maxillary advancement and bilateral sagittal split osteotomy to set back the mandible will camouflage the severity of the discrepancy without compromising the velopharyngeal function. In addition, patients who had pharyngoplasty or pharyngeal flap procedures in the past pose a certain limitation towards maxillary advancement and may require revision of soft tissue surgeries in order to prevent hypernasality. The other option is to use autologous costochondral bone graft in the maxillary gap following Lefort I advancement procedure. This results in better prognosis and stable treatment results [10].

Conclusion

a) soft palate length, width and Need’s ratio may vary with the underlying skeletal malocclusions

b) Males had increased soft palate length in skeletal class III malocclusion.

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Death Drive In Psychoanalysis versus Existential Psychotherapy-JuniperPublishers

Journal of Psychology and Behavioral Science-Juniper Publishers

Introduction

In Greek myth, the demon of death was the son of Nix (god of the night) and Erebus (god of darkness) and a twin to Hypnos (sleep). The term ‘death drive’ was coined by Freud for the first time 1920 in Beyond the Pleasure Principle, according to which 'Thanatos' is posited opposite of Eros, the creative and productive drives, sexuality and survival. In classic psychoanalytic theory the death drive (Thanatos) is seen as a drive towards selfdestruction and death. The death drive forces mankind into risky and self-destructive behaviors that could lead to death S Freud [1]. The death drive can be related to the work of the German philosopher Arthur Schopenhauer who in his book The World as Will and Representation by Schopenhauer A [2] says that we all exist by the will to live which is affirmed by pleasure. But he believed that life is a source of suffering and pain Heidegger [3]. Analysis of death is focusing on the existential significance and wants to understand the death phenomenon.

We need to understand and see Dasein as a whole, as a possibility. Dasein sees death as something inevitable but something that is happening to others and not to him. Therefore the concept of death is something that Dasein doesn't connect with the I Jasper, however, views Dasein as a way of being who, despite being part of existence is nevertheless impossible to understand merely as an isolated individual. According to Jaspers [4] human beings are aware that their future death is inevitable, but since they cannot experience their own death they do not perceive death as something to be concerned about. Others may hide from its existence by viewing it as belonging to some magical or nihilistic dimension. I choose to discuss the death drive in the context of psychoanalysis and existential psychotherapy.

Beyond the pleasure

In his book Project for a Scientific Psychology by S Freud [5] says that all mankind's mental events are directed and regulated by id which is the biggest part of the human mind and makes people to desire pleasure and avoid pain at any cost. In Civilisation and Its Discontents by S Freud [6] says that the pleasure principle determines the purpose of life thus being analogous to the reality principle. But then Freud realized that there were three conflicting facts in the human mind that he could not explain with the pleasure principle, and that led him to another principle which he found beyond the pleasure principle, and this in turn led him to the concept that later became known as the death drive.

The first conflicting problem Freud was confronted with was the paradox of PTSD in working with traumatized soldiers who had participated in World War I. He observed that they had this tendency to repeat or illustrate their traumatic experiences in a way that brought them back to the combat scene, and this contradicted Freud's pleasure principle or other people with odd behaviours that were engaged repeatedly in behaviours in which there is hard to find any source of pleasure

Another exception was discovered when Freud observed children's games e.g. when his grandson staged his mother's disappearance or when other children threw their toys away. This made Freud wonder how repetition of these distressing experiences would fit within the pleasure principle.

The repletion compulsion was the third exception. Freud had noticed in his practice that his patients when dealing with very distressing and painful life events would repress these experiences, but then they regularly felt compelled to repeat them, not as a memory from the past but as a current experience. The repetition compulsion in Freud’s eyes was a primitive instinct.

To seek other explanations for this destructive drive Freud revised his previous views on masochism which he had viewed as a consequence of traumatic childhood experiences. In The Ego and the Id by S Freud [7] he argues that the death drive expresses itself partially through destruction which is directed against the world. A year later he was more clear in his explanations and said that the libido's function is to make the destructive drive innocent by redirecting it outwards. The drive at this point was called the destructive drive, the drive for superiority, or the will power.

At the end of the decade in Civilization and Its Discontents he finally admitted that these new views had attained such a great hold upon him that he no longer could think in any other way. From a philosophical viewpoint the death instinct can be related to the German philosopher Schopenhauer's work. In his book The World as Will and Representation he said that all mankind exist by metaphysical powers, namely the “Will’ and that pleasure maintain this drive. But since he was a pessimist he believed that this confirmation of the will to live was an unfavorable and unethical thing, as he believed that life produces more pain than pleasure and bliss. The death instinct is a manifestation of an essential and common antithesis of the will power [8].

Heidegger's Analysis of Death

Heidegger's definition of death is not focusing upon people’s feelings when they are at the death stage nor is he concerned with death as a biological phenomenon. By facing death the meaning of being is defined and clear for 'Dasein'. In Heidegger's view there are two ways of being: authentic and inauthentic. Heidegger does not give an explanation for death itself but offers a phenomenology of our relationship to death. Even though his philosophy is thoughtful it is also gloomy. His account of death portrays a no-hope mode of being, and he has often been criticized for this [3]. Analysis of death is focusing on the existential significance and wants to understand the death phenomenon. We need to understand and see 'Dasein'as a whole, as a possibility. 'Dasein'sees death as something inevitable but something that is happening to others and not to himself. Therefore the concept of death is something that 'Dasein'doesn’t connect with the ‘I’.

'Dasein' understands the reality of death as an ever happening phenomenon in the world but it only happens to others and it has nothing to do with me and thus death continues to live unbeknownst to all and with no contact with the ‘I’. When ‘Dasein'faces his own death then that is a completely different phenomenon than facing other’s death. The fact that I die means the end of my potential, the end of my world. According to Heidegger the fear of my own death is in essence the fear of my extinction as a human being. This causes me a great deal of anxiety. I may be able to face other people’s death but may find it virtually impossible to come to terms with my own. 'Dasein'cannot experience its own death. As long as 'Dasein exists, it is not complete, that is, there are still some of its possibilities outstanding. If, however, 'Dasein'dies, then it is 'no- longer there’.

Jaspers' Analysis of Death

Jaspers [4] Talks about the opportunity for ‘Existenz'to the final phenomena, which is Transcendence, which is neither eternity nor total eradication. Even though Jaspers’ notion of death is not religious, he nevertheless uses some existential terms i.e. 'Existenz, Transcendence and Being that usually have religious connotation but under a different phraseology.

According to Dasein is a mode of being which manifests itself as the empirical self with a temporal dimension. It is a part of the world but cannot be understood as an object in isolation. 'Existenz', however, is the true mode of being this is an inevitable condition of man's existence. Moreover, there are four major 'boundary situations’ (those situations which threaten our sense of security and the foundation of our existence) of which the most important one is death because it signifies the end of man’s world.

According to human beings are aware that their future death is inevitable, but since they cannot experience their own death they do not perceive death as something to be concerned about. Others may hide from its existence by viewing it as belonging to some magical or nihilistic dimension Jaspers [3].

According to Jaspers [4] death has two different meanings. Death is perceived as either the cessation of existence as an objective fact, or as a specific boundary situation. Facing one's own death is a specific boundary situation and it is personal because Existenz convinces itself that Dasein- the basis of its empirical existence, i.e. the bodily existence - is temporal and transient and has to come to an end. Unlike the end of one’s empirical being, Existenz itself is not subject to death. As Existenz we are concerned with the significance of death and how we relate to it. We know that we have to face up to nothingness as there is no return for 'Dasein'and we will have to come to terms with this [4] says when we lose someone we love then life becomes an isolated existence for us, and as result of agony and bereavement we feel hopeless which could lead us to the pain and boundary situation of death. Even though death annihilates the person we love, the existential connection is intact and infinite. Based on man can understand the undeniable truth of his future death and the notion of annihilation.

Man believes that as long as he is alive he cannot experience his own death, and once he ceases to be alive he cannot experience it either - a typical Epicurean argument! So, the experience of one's own death does not seem possible. As a result, he does not perceive death as a reason for concern. He ignores his possible Existenz and clings on to his worldly activities. Alternatively, Dasein may ignore its everyday existence entirely and hide within its nihilistic or mystical realms. This would be another way of avoiding boundary situations. Thus, if man cannot face up to death existentially, he either preoccupies himself with worldly things or escapes into the mystical realms.

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A Correlative Study of Individualization from Hieroglyphics Documents among Siblings-JuniperPublishers

Journal of Forensic Sciences & Criminal Investigation-Juniper Publishers

Abstract

Questioned documents which consists handwritten documents, computerized printout, and symbol or sign used for communication among groups, has aroused and increasing with the passage of time. Yet, we are facilitated with advance technologies but still a few of the aspects are untouched. During the analysis of documents examination, we rely on the class and individual characteristics. It is considered that human is not a machine who can work similar, hence, a variation is encountered which is known as the natural variation. Every individual has their own individual characteristics, formation of letters, style of writing which may be adopted from the handwriting of someone else or might be adopted during the phase of maturation. This study was carried out to determine any similarity between the handwriting among siblings, because, it is considered that both might have learnt or adopted the same characteristics from same school, parents or from any group. During this study, it was observed that t-value between the handwriting characteristics of siblings was -1.4236 and the p-value was 0.0925 at (0.0925 <0.10). It shows that the handwriting of siblings is influenced from each other, teacher or parents and bear an elevated level of significance.

Keywords: Handwritten documents; Individuality; Characteristics; Siblings

Introduction

Handwriting is considered the reflection of a person's state of mind may reveal a lot his personality and mind. According to Huber, handwriting as a complex motor skill which is a combination of sensory, neurological and physiological impulses. Every person has a unique style of writing which usually remains similar throughout life time but addition of more individual characteristics continues. Individual characteristics are responsible factors which help to identify the writer or determine the similarity or distinguish between two different handwriting samples. Handwriting examination is based on the facts that no two persons can write exactly similar although some variations are always a part of their handwriting [1,2].

These variations are used to establish individuality among the unknown authors although some individual characteristics are believed to get inherited from one generation to another generation among the close genotype family members. The inherited traits within a family are taught or to write under similar circumstances by same parents and teachers. Moreover, if the both siblings fall under the similar age group then they also learn to write by imitating each other's writing habits which may be influenced by any particular writing style. Therefore, during the development of maturation of handwriting in early phase of life; similar characteristics are observed in most of the cases [3,4].

The handwritten documents vary in individual characteristics matric system and overall appearance that may be because of temperature, disease, age, health etc. Therefore, the individualization from handwritten documents among siblings and family members has a question against interpersonal and interpretational variation. Earlier in the 20th century, a few of the scientists comes out with study over twins, siblings and their correlation. In 1928, Kramer and his colleagues conducted a study on the handwriting resemblance between twins and siblings and come out with a significance result. According to that study, a greater degree of resemblance was observed in handwriting. Later in 1987, Munch reported the similarity between the handwriting of mother and daughter in form of a study.

In the same manner, Kiran P, Monica S conducted assorted studies on the determination of inheritance pattern and inherited traits between the family members. Following her earlier studies, Monica S conducted a study to find out the impact of heredity and environment in handwriting by using the computational features on MATLAB. As the resultant of that study, it was revealed that genetics has a role in shaping the writing habits of writer and handwriting is mixture of both nature and nurture. This study was carried out to determine any similarity among the individual characteristics in the handwriting of siblings and any significant correlation between their inherited traits of handwriting. For this study, the individual characteristics of handwriting such as formation of letters, initial stroke, connecting stroke, rhythm, pen lifting and tremors were studied. Because of this study, it was observed that the handwriting of the siblings resembles with each other due to similarity found in the individual characteristics. Though, the overall appearance of the handwriting might or might not be similar but when carefully examined the formation pattern of the letters is found to be similar in the handwriting samples of siblings [5,6].

Methodology

Handwritten documents consist characteristics of an individual style of writing adopted from the family members, teacher or may be adopted during the phase of maturation of development. As per the earlier discussion, this type of documents is encountered in fraudulent cases and the analysis of such documents leads towards the individualization of suspect. This study was carried out to determine and to establish any relation between the writings among siblings [7,8].

Material and Method

For this present study, a total of 50 samples were collected from 25 families which including male and female siblings from each family. All the samples were collected on A4 size sheet with the help of blue ball point pen. To take the samples, a standard letter was given to the subjects and was asked to copy of it. All samples were collected in the month of March- April 2017, when the temperature was in between 180-350 C. Simple random sampling method was used during the collection of samples. All the subjects were informed about the purpose of the study and prior consent of the subject was taken. All the collected samples were preserved in a white A4 size envelope and were placed at room temperature for further analysis process [9,10]. All the samples were manually examined by using stereoscope and hand lens of 10 X. All the inherited traits were examined based on the individual characteristics. For the photography of samples, a Vivo V-3 phone model with 13 mega-pixel camera quality was used. A hypothesis was set up for the analysis in this study to conclude the individuality of an individual based on hieroglyphics material [9-10]. The inherited traits might be similar among the siblings. Therefore, Hypothesis; H0; - siblings have similarity among the handwriting. H1; there is no correlation between the handwriting of siblings. Hence, H0 is not rejected in favor of H1 which represent that siblings have similarity in their handwriting.

Result & Discussion

This study was carried out to determine individuality in the handwriting characteristics among siblings (brother and sister) based on the individual characteristics. It is considered that siblings who study in same school, taught by same teacher and parents inherit some of the writing characteristics from them. Although during the phase of development, these features change and an individual adopt his/her own characteristics. Since, the siblings follow same trend though have possibilities of possession same writing skills and characteristics. In this study, some of individual characteristics i.e. formation of letters, initial strokes, connecting strokes, rhythm, pen lifting and tremors were examined in a few selected alphabets. These alphabets (A, O, M F, G, and Y) were selected from the written material by an individual that included; starting of words, middle letters and ending letter of any word.

Through the analysis process of handwritten material, it was observed that formation of letters was highly similar between the writings of siblings. It might be an effect of same educational system, influence of parent's handwriting etc. The observed similarities in the formation of letters and connecting strokes are given below in (Table 1). In the above table, the similarities between the writings of siblings were discussed. In which, the formation of letters, connecting strokes, line quality and other class characteristics were examined carefully that were highly influenced from the handwriting of each other. While in (Table 2) the individual characteristics of handwriting were examined. The observation of handwriting features i.e. formation, initial strokes, connecting strokes; rhythm, pen lifting and tremors were examined for both siblings (brother & sister) [9,10].

As a resultant of the study, it was found that the individual characteristics were highly discriminating but influenced from each other. The details are given below in the (Table 2). To determine any correlation between the handwriting of siblings, the correlation coefficient was examined. The correlation coefficient was obtained positive with the value of R-value 0.8328 and R2- value was 0.6936. The values are given below in (Table 3). This is positive correlation, which means that the characteristics of brother's handwriting have high positive values with his sister's handwriting characteristics. To determine the level of significance in among the handwriting of brother and sister, one tailed t-test was applied. The significance level was examined at values p< 0.10 which is given below in (Table 4).

From the above test of significance, it was observed that t-value between the characteristics was -1.4236 and the p-value was 0.0925. The obtained values of handwriting characteristics (0.0925 <0.10) is less than the significance level which satisfied the correlation between brother and sister. As per our earlier study, we observed that the writing of an individual shows the reflectance of their inherited traits of writing from parents while in this study, the siblings writing reflect the characteristics of similarity at very high level. This study can be helpful for the questioned documents examination especially in those cases where the specimen or admitted writings couldn’t be available. This study can be helpful to provide the clue and minimize the number of suspects involved in case. As an advance implementation of rhythmic system in handwriting examination has proved its significance, therefore, it can also improve its significance in the handwriting of the siblings and can put on par of other existing analysis procedures.

Conclusion

Handwriting of the siblings within a family resembles the influence of their parents writing, hence, it can be assumed that the handwriting of the siblings will also resemble from each other. It might be possible and a cause of that both had learnt the pre-writing skills under similar circumstances and might have imitated each other's handwriting. Due to this their writing style, it is quite possible that the writing characteristics may be found similar with each other. As the resultant of this study, it has been proved that sibling’s handwriting is influenced from the writing of each other at an elevated level. It can be a milestone in the field of forensic investigation to prove the authenticity of handwritten documents in fraudulent cases. If any kind of suspected document is recovered and the admitted or the specimen is not available in that case, the documents recovered from the family or siblings can be put up for the analysis of handwritten questioned documents.

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Oxidative Stress in Bacteria Measured by Flow Cytometry-JuniperPublishers

Journal of Biotechnology & Microbiology-Juniper Publishers

Abstract

The CellROX® Deep Red flow cytometry kit (Life technologies) has been developed for the detection of oxidative stress in mammalian cells. It combines a ROS sensitive fluorophore (CellROX® Deep Red) and a "viability” dye (SYTOX® Blue) to allow the detection of non-oxidized, oxidized and damaged cells. The present study investigated the application of these markers to Enterococcus faecalis and Fusobacterium nucleatum subjected to oxidative stress. An optimal concentration of CellROX® has been determined on Enterococcus faecalis and Fusobacterium nucleatum exposed to oxidative stress (H2O2). Bacteria have been exposed to various H2O2 concentrations and labeled with CellROX® to verify that fluorescence increased along with oxidative stress. Also, bacteria exposed to H2O2 were double stained with CellROX® and SYTOX® Blue and analyzed by flow cytometry. The optimal concentration of CellROX® Deep Red was 4^M for both strains. Fluorescence of bacteria labeled with 4^M of CellROX® Deep Red increased accordingly with the oxidative stress applied. Flow cytometry analysis of double stained samples showed bacteria subpopulations with increased CellROX® signal when stressed, and higher SYTOX® Blue uptake under higher oxidative stress. Results indicate that CellROX® Deep Red can be applied to measure oxidative stress in E. faecalis and F. nucleatum. The combination of CellROX® and SYTOX® Blue allowed the discrimination of non-oxidized, oxidized and damaged bacteria.

Keywords: Oxidative stress; ROS; Enterococcus faecalis; Fusobacterium nucleatum; CellROX™ Deep Red; Flow cytometry

Introduction

Free radicals are defined as highly reactive chemicals exhibiting unpaired electrons such as reactive oxygen species (ROS) [1]. Accumulation of ROS in cells damages nucleic and amino acids, membrane lipids and can initiate self-propagating oxidative chain reactions [2-5]. Developing methods able to correlate oxidative stress with bacterial response and survival would help to gain insight into the development of new antibacterial strategies. Unfortunately, the propensity of ROS to acquire electrons renders them highly reactive, short lived, and therefore very difficult to detect [6,7].

The use of ROS sensitive probes, that can be detected using several analytical methods such as spectrofluorometry, fluorescence microscopy or flow cytometry, offer high sensitivity and experimental convenience [8-10]. The ROS reporters hydroxy-phenyl-fluorescein and "singlet oxygen sensor green" have been previously employed to detect hydroxyl radicals and singlet oxygen in a cell-free model [11]. Dichlorofluoresceindiacetate and flow cytometry (FCM) were used by Subramanian et al. [12] to evaluate whether resveratrol, a redox active phytoalexin, can induce oxidative stress in E. coli [12]. However, the combined use of a ROS reporter with a "viability” marker to simultaneously assess oxidative stress and viability has never been investigated in bacteria.

The CellROX® Deep Red flow cytometry assay kit (Life technologies), recently developed to assess the effects of oxidative stress in mammalian cells, comprises two dyes, namely: the CellROX® Deep Red, a ROS reporter, and the SYTOX® Blue, a "viability” dye. CellROX® Deep Red is cell permeable, cytoplasmic and does not fluoresce in a reduced state, while it becomes fluorescent upon oxidation. The second dye, SYTOX® Blue, is a cyanine nucleic acid stain which only diffuses into membrane-damaged cells. Only one epifluorescence microscopy study has used the CellROX® Deep Red to visualize oxidative stress in ionophore-treated Bacillus subtilis, whereas SYTOX® Blue has been previously used to assess bacterial membrane integrity by flow cytometry [13-15].

However, flow cytometry (FCM) has never been applied to correlate oxidative stress and viability in bacteria using the combination of these two dyes. The aim of this study was to assess the ability of the CellROX® Deep Red dye to detect oxidative stress in bacteria and to monitor membrane integrity using SYTOX® Blue. Specifically, FCM was used to assess the effect of various concentrations of hydrogen peroxide on Enterococcus faecalis and Fusobacterium nucleatum. The capacity of the combination of CellROX® Deep Red and SYTOX® Blue to distinguish non-oxidized (CellROX® Deep Red negative cells) from oxidized (CellROX® Deep Red positive cells) and membrane-damaged bacteria (SYTOX® Blue positive cells) was further investigated.

Material and Methods

Bacteria

A Gram-positive bacterium, Enterococcus faecalis (E. faecalis 135737, culture collection of the University Hospitals of Geneva, CH) and a Gram-negative bacterium, Fusobacterium nucleatum (Orale Mikrobiolgie Zurich culture collection - OMZ 598) have been used in this study. E. faecalis and F nucleatum were cultured from frozen stocks onto Columbia and Schaedler agar plates respectively. Bacteria from agar cultures were transferred in liquid media and incubated overnight at 37 °C (stationary phase). Brain heart infusion has been used as liquid media for E. faecalis and Schadler broth for F. nucleatum (all from Oxoid AG, Pratteln, Switzerland). Cultures of F. nucleatum were maintained under anaerobic conditions using packs of carbon dioxide (GasPack Anaerobe Gas Generating Pouch system with indicator, Becton Dickinson). On the day of the experiment bacteria were centrifuged and re-suspended in NaCl 0.9% to spectrophotometrically adjust the bacterial concentration at OD,600nm 0.2: ~1.2 x107 bacteria, Biowave II,’ Biochrom WPA, Cambridge, GB).

Viability curve

Hydrogen peroxide 3% w/w (Bichsel AG, Interlaken, Bern, Switzerland) has been used to induce oxidative stress in bacteria. To ensure that oxidative stress was measured on live cells, a H2O2 dose-response was determined for both strains using the LIVE/DEAD BacLight Bacterial Viability kit (Life technologies, Switzerland) and the Accuri C6 flow cytometer as previously described (BD Accuri Cytometers, Ann Arbor, USA) [16,17]. Briefly, 100μL of bacterial suspension have been mixed with 100μL of hydrogen peroxide at different concentrations (from 0 to 980mM in samples) and incubated at 37 °C for 15min before staining with the LIVE/DEAD solution. A sublethal range of H2O2 was defined as concentrations causing less than 10% cell death.

Defining a labeling concentration of CellROX® Deep Red for bacteria

To define an optimal labeling concentration of CellROX® Deep Red (Life technologies, Switzerland) for E. faecalis and F.nucleatum, bacteria have been exposed to maximal sublethal H2O2 concentrations (as defined by the viability curve), during 15min at 37 °C. Cultures were then incubated with various concentrations of CellROX® Deep Red (0μM, 0.5μM, 1μM, 2μM, 4μM and 8μM) during 30min. After incubation, bacteria were sonicated 20sec to disperse aggregates (35kHz, Sonoroex, Bandelin electronics, Berlin, DE) and fixed with 3.7% formaldehyde (VWR International AG, Dietikon, Switzerland). Fixation has been performed for biosafety reasons.

Testing increasing H2O2concentrations

To verify that the CellROX® Deep Red fluorescence increased accordingly with oxidative stress, bacterial samples were exposed to different concentrations of H2O2 (from 0mM to 160mM in samples) for 15min at 37 °C. The samples have then been incubated with 4μM CellROX® Deep Red (previously determined working concentration) during 30min at 37 °C, sonicated 20sec and formaldehyde fixed. Control samples were labelled with 4μM CellROX® Deep Red without prior exposure to H2O2.

Dual labeling with CellROX® Deep Red and SYTOX® Blue

To test whether the combination of CellROX® Deep Red and SYTOX® Blue would identify different bacterial oxidative states, samples of E. faecalis and F. nucleatum have been exposed for 15min to different concentrations of H2O2 (0mM- 980mM in samples) and then incubated with 4μM CellROX® Deep Red for 30min at 37 °C. Controls including non-oxidized (0mM H2O2), oxidized (maximum sublethal H2O2 concentration) and membrane-damaged (980mM H2O2) cells were used for comparisons. Samples were then sonicated 20sec, and SYTOX® Blue added to a final concentration of 4μM. Incubation with the SYTOX® Blue was continued for 15min. Experiments using both CellROX® Deep Red and SYTOX® Blue have been made on live cells (no formaldehyde fixation).

Flow cytometric analysis

Samples have been analyzed using a Gallios flow cytometer (Beckman Coulter, California, USA). Prior to analysis, unlabeled bacteria were run to optimize voltage settings (trigger on the FSC, voltage 650 on the FL-6 channel, 500 on the FL-9), and flow rate was set on low. Bacterial events were discriminated from debris using forward (FSC-A) and side scatter (SSC-A). Doublets have been excluded for analysis by FSC-height and width. CellROX® Deep Red signal (excitation/emission; 644/665) was collected in the FL6 channel (BP 660/20) and the SYTOX® Blue signal (excitation/emission; 444/480) in the FL9 channel (BP 450/50). Gates applied for population discrimination were set manually based on control samples. Data were exported and analyzed with FlowJo software (FlowJo for Windows, version 10.0.06, 2014, Tree Star Inc., Ashland, Oregon, U.S.A.). The geometric mean of fluorescence intensities (MFI) was expressed as relative fluorescence units (RFU).

Statistical analysis

All experiments were performed in triplicate and repeated 3 times. Results were statistically analyzed using one-way analysis of variance (ANOVA) and Tukey multiple comparison intervals (α = 0.05).

Results

Viability curve

Concentrations of H2O2 up to 80mM and 40mM were respectively applied on E. faecalis and F. nucleatum without inducing more than 10% reduction in viability (data not shown). Therefore, these concentrations were defined as the maximum oxidative stress to be applied on E. faecalis and F nucleatum, and were retained to determine an optimum working concentration of CellROX® Deep Red for measuring oxidative stress.

Defining a labeling concentration of CellROX® Deep Red for bacteria

Control suspensions (0μM CellROX® Deep Red) of E. faecalis and F nucleatum respectively exposed to 80mM and 40mM H2O2 displayed a natural fluorescence around 300 RFU (Figure 1A & 1B). Concentrations of CellROX® Deep Red ranging from 0.5μM to 4μM resulted in increased fluorescence intensities in both bacterial species. At 4μM CellROX® Deep Red, fluorescence intensities reached 2807±945 RFU and 3104±1151 RFU for E. faecalis and F. nucleatum respectively. Further increasing CellROX® Deep Red concentrations to 8μM resulted in decreased fluorescence intensities, for both strains (Figure 1A & 1B). Therefore, 4μM has been selected as the optimal concentration of CellROX® Deep Red for measuring oxidative stress in E. faecalis and F. nucleatum.

Testing increasing H2O2 concentrations

Control cultures (0mM H2O2) labeled with 4μM CellROX® Deep Red displayed a fluorescence of 648±90 RFU for E. faecalis and 1138±396 RFU for F. nucleatum (Figure 1C & 1D). For both strains, increasing concentrations of H2O2 progressively increased CellROX® Deep Red signal. For E. faecalis, the fluorescence signal was maximal at 80mM H2O2 (3001±633 RFU), whereas F nucleatum exhibited maximal fluorescence intensities at 40mM H2O2 (3753±1575 RFU) (Figure 1C & 1D). These H2O2 concentrations corresponded to the maximum sublethal values of each strain. Increasing H2O2 concentrations above these values resulted in decreased CellROX® Deep Red fluorescence in both strains.

Dual labeling with CellROX® Deep Red and SYTOX® Blue

Flow cytometric analysis of non-oxidized controls (0mM H2O2) showed 96.2% of E. faecalis and 85.6% of F. nucleatum with low signals for both CellROX® Deep Red and SYTOX® Blue (Figure 2A & 2E). Oxidized controls (samples under maximal H2O2 stress) exhibited increased CellROX® Deep Red signal (Figure 2B & 2F). At 160mM H2O2, bacteria of both strains displayed increased SYTOX® Blue signal (Figure 2C & 2G). When exposed to 980mM H2O2, 97.1% of E. faecalis and 98.8% of F. nucleatum exhibited a SYTOX® Blue positive signal in addition to the CellROX® Deep Red fluorescence (Figure 2D & 2H). Notably, CellROX® Deep Red signal intensity decreased in E. faecalis at H2O2 concentrations above 80 mM (Figure 2B-2D).

Discussion

In the current study, we successfully applied both the CellROX® Deep Red and the SYTOX® Blue to a Gram-positive and -negative bacterium to measure oxidative stress and to discriminate non-oxidized from oxidized and damaged cells.

Concentrations of hydrogen peroxide selected to induce oxidative stress were in the millimolar range (40mM-80mM). Previous studies have shown that E. faecalis exposed 15min to H2O2 retains viability up to 15-20mM [18,19]. The higher concentrations reported in the current study may be attributed to the different methods used for viability measurement. Whereas FCM in combination with the LIVE/DEAD staining has been used in this study to evaluate membrane integrity, previous studies measured viability by culture plating techniques [20]. There is evidence showing that bacteria proliferation capacity is affected at lower concentrations of H2O2 than membrane integrity [21-23].

An optimal concentration of CellROX® Deep Red has been determined by testing several concentrations of dye on stressed bacteria. Results indicate that fluorescence increased when increasing concentrations of dye up to 4μM, but tended to decrease at 8μM (Figure 1A & 1B). This pattern of signal intensity suggests equilibrium between the dye and oxidants. At concentrations below 4μM, the dye would be saturated by oxidants and additional oxidative stress would not be detected by lack of free dye. Therefore CellROX® Deep Red at 4μM has been selected as the optimal labeling concentration in these bacterial strains. The tendency of fluorescence to decrease at concentrations above 4μM, may be explained by a phenomenon of FRET quenching since the excitation/emission spectra of the CellROX® Deep Red overlap (644/665nm) [24].

Samples labeled with 4μM CellROX® Deep Red and exposed to increasing oxidative stress displayed gradually increasing fluorescence signals with highest intensities at the maximal sublethal H2O2 concentrations. These data confirm that CellROX® Deep Red fluorescence increased accordingly with the oxidative stress applied. Notably, F nucleatum exhibited higher fluorescence intensities than E. faecalis at all H2O2 concentrations tested (Figure 1C & 1D). A possible explanation might be related to a higher uptake capacity of CellROX® Deep Red by F. nucleatum, since the bacterium has a larger cytoplasm; E. faecalis measures about 1μm while F. nucleatum reaches 5-25μm [25,26]. Also strict anaerobes as F. nucleatum, are more sensitive to oxidative stress probably due to enzymes particularly prone to oxidation, as those containing exposed glycyl radicals and low-potential iron sulfur clusters [27,28]. Reaction with these targets may lead to further oxidative reactions potentially responsible for higher ROS production and fluorescence signal in F. nucleatum.

The combination of CellROX® Deep Red and SYTOX® Blue showed that control cells (0mM H2O2) displayed a double negative signal and were located in quadrant 4 (Figure 2A & 2E). On the contrary, oxidized controls (40mM and 80mM) displayed an increased CellROX® Deep Red signal, testimony of oxidative stress; these bacteria were located in quadrant 3 (Figure 2B & 2F). For both strains an uptake of SYTOX® Blue at H2O2 concentrations above sub lethal values (160mM) was observed, thereby confirming that membrane injury occurred under such elevated oxidative stress (Figure 2C & 2G). In E. faecalis, sub-populations characterized by different intracellular concentrations of dyes were observed, possibly indicating intermediate states of membrane injury (Figure 2C). Membrane- damaged controls (980mM) showed bacteria positive for both CellROX® Deep Red and SYTOX® Blue (Figure 2D & 2H). This is not surprising as the membrane-damage of these cells was produced by an excess of oxidative stress. However, E. faecalis cells lost CellROX® Deep Red fluorescence possibly due to a leakage of the dye from the cytoplasm, since membrane injury was shown to occur at such elevated H2O2 concentrations (Figure 2B-2D). The presence of an outer membrane in the Gram-negative bacterium, F. nucleatum, may have accounted for a better cytoplasmic retention of the CellROX® Deep Red dye. Gram-positive and -negative bateria have previously been shown to react differently to the same staining protocol [29].

Conclusion

The results of the current study indicate that the CellROX® Deep Red dye can be used to measure oxidative stress in E. faecalis and F. nucleatum. The combinational use of CellROX® Deep Red and SYTOX® Blue allowed the identification of nonoxidized, oxidized and damaged bacteria. Future studies seem warranted to assess the reactivity of the CellROX® Deep Red dye in presence of other oxidative challenges.

Acknowledgments

This study was supported by Grant #31003A-149962 of the Swiss National Science foundation. Mr. Aubry- Lachainaye JP and Mrs. Gamerio C are acknowledged for their contribution during flow cytrometry analysis. The authors deny any conflicts of interest related to this study.

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