P < 0·001) and 48·8 % did not consume fruits on a daily basis. Moreover, 39·1 % did not engage in physical activity, and over 35 % spent more than 5 h/d on screens. A significant association between the frequency of training during the pandemic and the reported change in weight was found (P < 0·001). A significantly higher percentage of participants reported physical and emotional exhaustion, irritability and tension either all the time or a large part of the time during the pandemic (P < 0·001). Although a high percentage of participants reported sleeping more hours per night during the pandemic, 63 % had sleep disturbances. The study highlights that the lockdown due to the COVID-19 pandemic caused a variety of lifestyle changes, physical inactivity and psychological problems among adults in the MENA region.Coronavirus disease 2019 (COVID-19) has rapidly spread globally, forcing countries to apply lockdowns and strict social distancing measures. The aim of this study was to assess eating habits and lifestyle behaviours among residents of the Middle East and North Africa (MENA) region during the lockdown. A cross-sectional study among adult residents of the MENA region was conducted using an online questionnaire designed on Google Forms during April 2020. A total of 2970 participants from eighteen countries participated in the present study. During the pandemic, over 30 % reported weight gain, 6·2 % consumed five or more meals per d compared with 2·2 % before the pandemic ( Despite strategies adopted by the Chinese government to stop the infection, it continued to spread throughout the world. By the end of January 2020, WHO declared COVID-19 as a public health emergency of international concern and on 11 March 2020, WHO characterised this epidemiological phenomenon as a global pandemic. According to the situation report published by the WHO on 5 July 2020, there were over 11 million confirmed cases globally and about 1·1 million cases in the Eastern Mediterranean Region. In the Middle East and North Africa (MENA) region, the Gulf countries like Saudi Arabia, Qatar, United Arab Emirates and Kuwait reported the highest numbers of confirmed cases proportionally to the population size. According to the Organization for Economic Cooperation and Development, some countries in the MENA region have taken crucial measures to combat this pandemic, closing schools, kindergartens, religious places, airports and malls, as well as preventing social gatherings. Others have gone far by suspending government departments.The outbreak of the 2019 novel coronavirus disease (COVID-19) was first reported in late December 2019 solely in the city of Wuhan, China. Following the footsteps of China and the recommendations of the WHO, governments around the globe started implementing isolation, quarantine, social distancing and community containment at different magnitudes in order to reduce the spread of the virus and lessen its impact on medical resources,7.With more knowledge acquired on the virus, its rate of transmission, resemblance to previous outbreaks and the uncontrolled spread of the virus, governments worldwide had to act quickly. People were forced to stay at home, practice remote working and online learning from home and adhere with physical distancing. However, initiating such sudden changes proposes major modifications in the lifestyle and behaviours of the population. Personal restrictions might lead to physical inactivity especially in courtiers with complete lockdowns, like Jordan. Emotional stress and irritability are common psychological impacts during such times of crisis. A recent review on the psychological impact of quarantine reported a negative effect, and the stressor factors included long quarantine period, fear of infection, frustration, boredom, financial loss, stigma, inadequate supplies of goods and misinformation. Consequently, boredom and stress have been associated with overeating and greater energy intake mostly from comfort foods which are usually high in fat, sugar, salt and energy content,14. Quarantine-related stress might also cause sleep disturbances, which in turn has been linked to increased food intake and weight gain.In most countries, quarantine and physical distancing were initiated, school closures were mandatory and large gatherings were cancelled.To date, there is no specific cure or anti-viral medicine to treat COVID-19; therefore, maintaining healthy nutritional habits, following food safety recommendations, regular physical activity, coping with stress and adequate sleeping hours are of utmost importance during quarantineDespite the global administration of quarantine, little is known about its effect on the daily life of the general population in the MENA region. Therefore, the objective of this study was to assess eating habits and other lifestyle behaviours among residents of the MENA region after implementing lockdown and travel restrictions. Moreover, understanding the effect quarantine has on eating habits and lifestyle on individuals could be proved essential in designing intervention protocols in the possibility of another lockdown caused by a new wave of the same or mutated virulent virus.This cross-sectional, online survey was conducted in the Greater Middle East region between 15 April 2020 and 29 April 2020. The sample was drawn from eighteen countries within the MENA region, including Algeria, Bahrain, Egypt, Iraq, Jordan, Kuwait, Lebanon, Libya, Morocco, Oman, Palestine, Qatar, Saudi Arabia, Sudan, Syria, Tunisia, United Arab Emirates and Yemen. Adults aged 18 years and older were recruited in the study using a convenience and snowball sampling method. This method depends on data collection from population members who are conveniently available to participate in the study. There was no restriction on the total number of participants; however, we aimed at a minimum of 100 participants from each country., the International Physical Activity Questionnaire Short Form and the Copenhagen Psychosocial Questionnaire. The questions were then translated to Arabic and French and culturally adapted following internationally accepted methodology,21. The questionnaire was then reviewed by other members of the research team and was pilot-tested with thirty people from five countries in the MENA region. Minor modifications in wording were made to the questionnaire following the pilot-testing. The questionnaire that included fifty-eight questions was divided into seven sections: (1) socio-demographic background (thirteen items): age, sex, country of residence, education level, employment status, marital status, number of children, height, weight, health status, a work or study setting; (2) sources of information (two items): source of health-related information and source of nutrition-related information; (3) eating habits (nineteen items): meal type, meal frequency, eating breakfast, skipping meals, water intake, food choices, changes in food intake, following a diet, taking supplements and consuming herbs; (4) shopping habits (six items): using a grocery list, stocking up, online shopping, reading food labels and cleaning/sanitising groceries; (5) physical activity (eight items): exercising frequency and duration, household chores frequency and duration and sedentary time for work, study and entertainment; (6) stress and irritability (two items): physical exhaustion, emotional exhaustion, irritability and tension and (7) sleep (eight items) sleep duration, sleep quality, sleep disturbances and energy level. The full version of the questionnaire is available in online Supplementary Appendix 1.A multicomponent, self-administrated online questionnaire was designed using Google Forms in English, Arabic and French. Two researchers from the College of Health Sciences at the University of Sharjah (United Arab Emirates) and the College of Food and Agriculture at United Arab Emirates University (United Arab Emirates) developed the initial draft of the questionnaire in English. The questions were developed based on the Short FFQThe survey was distributed through emails and social media platforms. An information sheet and consent form appeared on the first page of the survey indicating the study description, main objective and the right of participants to withdraw at any time. The consenting participants then chose their desired language of communication and proceeded to complete and submit the questionnaire. All data were collected anonymously, and no incentives were given for completing the questionnaire.This study was conducted according to the guidelines laid down in the Declaration of Helsinki, and all procedures involving human subjects were approved by the Research Ethics Committee at the University of Sharjah (REC-20-04-25-02) and the Social Sciences Research Ethics Committee at the United Arab Emirates University (ERS_2020_6106). Electronic informed consent was obtained from all participants.χ2 test was used to examine group differences for single observations in categorical variables, and McNemar’s test was used to investigate the difference between categorical variables before and during the COVID-19 pandemic (accounting for dependent observations). Results were significant for P value < 0·05. Statistical analysis was performed using Statistical Package for the Social Sciences version 26.0 (IBM).All variables presented in this study are of categorical nature since they represent population characteristics. Categorical variables are presented as frequencies and percentages . The The questionnaire was completed by 2970 participants. Most of them completed the survey in Arabic language (63·1 %) followed by English (33·8 %) and French (3 %). The demographic characteristics of the study population are presented in The male:female ratio was almost 1:3, with 28·4 % males. The majority of surveyed individuals were aged 18–25 years (29·6 %), were married (53·2 %), had no children (49·7 %), completed a university degree (48·5 %), worked full-time (44·8 %) and were working/studying from home during the lockdown (56·0 %). About one-third of the surveyed individuals reported weight gain during the COVID-19 pandemic (30·3 %), 16·9 % lost weight, 43·9 % maintained weight and 8·8 % did not know if there was a change in their weights. When asked to describe the general state of their health during the outbreak, the majority of surveyed individuals reported very good health state (37·0 %) and only 0·9 % reported poor state of health.Eighteen countries from the MENA region  participated in the survey. The largest proportion of respondents was from the United Arab Emirates (13·1 %), Jordan (11·9 %), Lebanon (11·5 %), Saudi Arabia (8·3 %) and Palestine (6·4 %).P < 0·001). Moreover, the percentage of participants skipping meals decreased from 64·4 % before the pandemic to 45·1 % during the pandemic (P < 0·001). Among those who reported skipping meals, 60·8 % stated that lack of time was the main reason before the pandemic and 37·9 % stated that lack of appetite was the main reason for skipping meals during the pandemic. Although the results showed an increase in meal frequency, 74·0 % of participants reported not meeting the recommended water intake by drinking less than eight cups of water per d during the pandemic.The results on eating habits  showed aMore than two-thirds of the respondents (65·7 %) sanitised their groceries before storage. Moreover, 76·4 % of participants reported preparing a list before grocery shopping. Online shopping was less common among the surveyed participants as only one-third reported ordering groceries online .P < 0·001). There was a significant association between the frequency of training during the pandemic and the reported change in weight (P < 0·001). Of those who reported training more than three times per week, 25·1 % lost weight and 48·9 % maintained their weight (P < 0·001). However, 36·6 % of people who did not train reported gaining weight.Data about frequency of physical activity among surveyed population are presented in P < 0·001). Moreover, the percentage of participants spending more than 5 h/d on screens for entertainment increased from 14·6 % before the pandemic to 37·5 % during the pandemic (P < 0·001)  .P < 0·001)  .P < 0·001). Moreover, a higher percentage of participants reported sleep disturbances during the pandemic (63·2 %) compared with before (53·1 %). As a result, 29·6 % of the participants reported feeling lazy and less energised during the pandemic, compared with only 4·7 % before the pandemic (P < 0·001)  .This cross-sectional study provides a snapshot of the eating habits and lifestyle factors for a sample of 2970 MENA adult residents, who answered an online survey in April 2020, approximately 7–8 weeks following the lockdown implementation to control the spread of the COVID-19 pandemic.. During the COVID-19 outbreak, misinformation overload (‘infodemic’) has bombarded social media, which might have harmed the mental health of individuals. A study has shown that 82·0 % of participants were regularly exposed to social media, and the increase of exposure frequency was found associated with higher odds of anxiety and depression. Thus, public awareness should be raised about reliable sources of information, especially during such crises.The results of this study showed that about two-thirds of the participants obtained their health- and nutrition-related information mainly through social media applications followed by credible websites of national and international health organisations and healthcare professionals. Similarly, a study in Iran reported that 55·3 % of surveyed nurses obtained their COVID-19 updates and information from the website of the WHO and the Ministry of Health, followed by 48·2 % who relied on social media applications as a main source of information.An increase in the number of meals consumed per d and a reduction in the percentage of skipping meals during the COVID-19 pandemic were reported by the participants in the present study. This also explains the reported weight gain among this population. Likewise, a survey among French adults cohort revealed unfavourable nutritional behaviours of weight gain, decreased physical activity, increased sedentary time, increased snacking, decreased consumption of fresh fruits and vegetables and increased consumption of sweets, biscuits and cakes. Likewise, in Italy, 86·6 % of surveyed participants reported drinking <2 litres of water per d during the COVID-19 pandemic and 26·2 % consumed <1 litre of water per d.About three-quarters of the participants in this study reported drinking less than eight cups of water per d during the COVID-19 pandemic, out of which about a third reported drinking <1 litre/d. These quantities do not meet the recommended water intake set by the WHO of 2·9 litres/d for males and 2·2 litres/d for females.Nearly half of the surveyed participants in this study did not consume fruits daily and one-third did not consume vegetables daily. On the other hand, one-third of the same population reported consuming sweets and salty snacks at least once per d. This unfavourable trend towards a Westernised diet was reported in an ecological study in the MENA region (1961–2007), as the proportion of energy derived from meat and vegetable oils increased significantly, while that from cereals, vegetables, fruits, milk and dairy products showed a descending trend,30. A recent meta-analysis of observational studies suggested that consuming fruits and vegetables is negatively associated with the metabolic syndrome and its risk factors. In addition to being a rich source of fibre, as well as various micronutrients and antioxidants, an adequate intake of fruits and vegetables might optimise the immunocompetence, a role indicated in both the prevention and treatment of COVID-19.The results clearly demonstrate the need for dietary support of individuals during lockdowns focusing on healthy eating choices. Consuming a diet rich in vegetables and fruits is especially important during these times due to their high content of antioxidants, phytonutrients and anti-inflammatory substances. Fortunately, only 14·1 % of study participants did not clean groceries before storage. Likewise, a study in India reported an increase in the frequency of using sanitisers, hand washing and wearing masks among the study participants. These findings indicate the increasing awareness of participants towards personal hygienic measures to avoid COVID-19 infection.Improving hygiene practices, cleaning groceries before storage and limiting grocery visits have been recommended by various health authorities globally during the COVID-19 pandemic,36. These findings further explain the weight gain witnessed in this study. Obesity rates in the MENA region are already alarmingly high and are associated with non-communicable diseases such as type 2 diabetes, CHD and stroke. The unprecedented self-quarantine mandate during the COVID-19 pandemic might have worsened this health problem not only among adults but also among children,39. Studies have indicated that physical inactivity results in changes to body composition  associated with increased insulin resistance, reduced cardiorespiratory fitness and increased dyslipidaemia,41. These changes can occur in as little as 2 weeks of physical inactivity and are reversible in younger people, but less so in older adults. A recent review suggested similar metabolic consequences during the COVID-19 confinement due to physical inactivity and overeating. The review recommended nutritional interventions and resistance training as potential strategies to prevent such deleterious metabolic effects. It is challenging, however, to exercise during lockdown when gyms and parks are closed and people are stressed, depressed and isolated,45. Awareness about different types of home exercises  and their benefits on weight status and mental health is essential,47.In this study, more than one-third of the participants reported not engaging in any physical activity during the COVID-19 pandemic and of those, over one-third reported gaining weight. Recent studies have shown an association between COVID-19 severity and mortality with increased weight and BMI categories among other co-morbidities. Our study found an increase in sleep hours during the pandemic compared with before the pandemic. Moreover, sleep disturbances have been reported by over 60 % of participants, and about 30 % of them had poor sleep quality during the pandemic. Similarly, a survey in Italy reported an increase in the percentage of participants sleeping 7–9 h per night from 49·9 % before the pandemic to 54·8 % during the pandemic. In addition, a survey in China indicated that 18·2 % of participants had poor sleep quality, and the percentage increased to about 24 % among healthcare workers.During the COVID-19 pandemic, people are exposed to a stressful situation for an unknown duration of time. This could disrupt sleep quality, which has a direct effect on the physical functioning during the subsequent day,51. A recent review highlighted four different mechanisms that stress can be linked to overweight and obesity including: (1) cognitive processes such as self-regulation; (2) behavioural effect through inducing overeating, shortening of sleep and decreasing physical activity; (3) physiological changes in the hypothalamic–pituitary–adrenal axis and (4) stimulating the production of biochemical hormones and peptides such as leptin, ghrelin and neuropeptide Y. Additionally, the main reason for skipping meals among participants in the present study before the pandemic was due to lack of time; however, during the pandemic, participants skipped meals mainly due to lack of appetite, which is common in the presence of depression. The results of our study indicate an increase in the percentage of participants reporting physical and emotional exhaustion, irritability and tension all the time, or a large part of the time during the COVID-19 pandemic.The COVID-19 pandemic has been associated with increased anxiety and distress, which in turn affects the lifestyle of individuals. The number of confirmed COVID-19 cases by the end of April widely varied between MENA countries and ranged between 21 000 cases in Saudi Arabia and six cases in Yemen. Countries like Saudi Arabia, United Arab Emirates and Qatar had over 10 000 confirmed cases by the end of April. However, Jordan, Palestine, Syria, Yemen, Libya and Sudan had <500 cases. The number of deaths also varied. United Arab Emirates, Jordan, Lebanon, Palestine, Bahrain, Sudan, Iraq, Syria, Kuwait, Oman, Tunisia, Yemen, Libya and Qatar had <100 deaths by the end of April, while Morocco, Egypt, Algeria and Saudi Arabia reported over 150 deaths in the same period. Although there was a variation in the number of cases and deaths between MENA countries, all countries included in the survey implemented strict quarantine measures between mid/end of March 2020 until June 2020 or even longer. Countries like Saudi Arabia, Bahrain, Sudan, Egypt and United Arab Emirate did not start easing restrictions until the end of June, while Yemen announced easing lockdown restrictions in mid-July. Restrictions included closure of boarders, closure of non-essential businesses, nightly travel curfew, local movement and travel restrictions, facilitating remote working and online learning, and cancelling prayers to avoid mass gathering events. Considering different stages of the pandemic in different MENA countries, the survey might not reflect such as diverse behaviour of the entire population from those places.This study was conducted during April 2020. By that time, all countries included in the survey have already declared the state of emergency due to COVID-19It is acknowledged that this study has several limitations, including the use of a self-reported questionnaire which might cause some respondent bias or misreporting of data; the cross-sectional study design which does not allow for causality evaluation and the use of convenience sample which limits generalisability of the results; although the aim was to get a minimum of 100 participants from each country, six countries had fewer participants. Also, the information on infection with COVID-19 and medical history was not determined in the study. Such analysis would not only require a longer questionnaire, which might have decreased the compliance and response rate, but also would have required a larger sample size based on the prevalence of all the factors to acquire adequate study power. Another potential limitation of the study might be a recency bias which suggests that recent habits are more easily retrieved from memory; however, data were collected only 1 month after lockdown to minimise memory failure for previous habits. The study information was acquired after lockdown, and although comparisons are critical to be made to draw inferences, no conclusive remarks can be drawn. Nevertheless, developing a web survey allowed a rapid assessment of eating and lifestyle habits in a very critical period of the pandemic and from around eighteen countries in the MENA region. It also facilitated the anonymity of the respondents, thus reducing the social desirability bias. The present study provides unique information about unprecedented circumstances that have faced mankind in recent history.The results of the present study suggest that the COVID-19 pandemic triggered a variety of unhealthy lifestyle changes, physical inactivity and psychological problems in the MENA region. There is also substantial need to increase awareness regarding healthy nutritional habits, general safety measures, importance of home-based physical activity and stress relief mechanisms. Finally, the provision of reliable health and nutrition information is essential as well as psychological support during the pandemic.


Background: The study was conducted to determine the bacterial composition and antibiotic susceptibility profiles of a research institute at the University of Ghana where workers and students spend about 70-85% of their lives in indoor and immediate-outdoor environments. This is imperative as one-third of the recognized infectious diseases are transmitted through airborne-route. Furthermore, the increasing rate of bacterial antimicrobial resistance associated with such environments poses serious public health challenges.Methods: A total of 42 airborne samples were collected from eight major sites at the Department of Biochemistry, Cell and Molecular Biology (BCMB), using passive bacterial sampling techniques. Standard phenotypic microbiological procedures were used to characterize the isolates. Antibiotic susceptibility profiles were determined using standard disk diffusion method and guidelines of Clinical and Laboratory Standards Institute (CLSI).Results: Four groups of bacterial isolates were identified from the total samples collected with Gram positive bacilli as the most common. All the isolates showed resistance to beta lactam and sulfonamide classes of antibiotics with full resistance (100%) to ampicillin and penicillin. In total, seven different anti-biotypes were observed with the highest susceptibility displayed towards tetracycline and gentamycin. Significantly, the various air sampling sites of the institute indicated the presence of bacteria with the majority showing multiple antibiotics resistance.Conclusions: Although the recovery of bacteria from supposed sterile environments calls for attention, the observed low contamination rate as compared to the WHO standard suggests a minimum risk of exposure of students and workers to airborne microbial contamination. Microorganisms are ubiquitous; they normally inhabit indoor and outdoor environments. The inhaled air in the indoor environment is dominated by a number of microorganisms, with consequent effects on the health those indoors2. Little is known about the diverse communities of bacteria shared by indoor environments such as houses, offices, laboratories, schools, hospitals, and other indoor environments where people work, relax or find solace4. The diversity of these microbes in the indoor environment is influenced by several factors such as water, temperature, moisturized surfaces or worktops, the rate of particle deposition, and other parameters like indoor pollutants, especially those generated by various human activities6.Quality of air, especially in indoor environments where people spend 80–95% of their lives is of significant health importance8. The presence of these biological contaminants has been reported in the air of hospitals9, but little is known about their impact on a typical research environment in Ghana10. As dangerous as bioaerosols are by themselves, they also secrete toxins that are transmitted by the airborne route through the nasal airways making indoor environment a potential source of human pathogens11. Microorganisms gain access to different indoor compartments of buildings through openings like doors, windows, blowing fan blades, air conditioners and the immediate-outdoor environments12. Immediate-outdoors areas, usual described as 0.9m to 3.5 away from the main building, includes foyers, such as the corridor, lobby or hallway13. In addition the indoor air microbiomes originating from outdoor air-space drifts14 have influencing factors which account for diverse microbial distribution. In a typical research environment of academic training and learning, a series of movements do occur from the outdoors through the immediate-outdoors to indoors14. This facilitates the movement of microorganisms, especially bacteria to different compartments of the building15.Bioaerosols, mostly bacterial and fungal spores are actively living complex particles that have been associated with contamination of indoor air12. Although most of these microbes have been reported as opportunistic pathogens, they are not necessarily associated with severe infections16. However, they can pose significant challenges to immune-compromised individuals18. Sterile conditions, especially in biological laboratories control microbial growth19. Interestingly, the microbes are able to survive using the air routes to other favourable environments within indoor environments15. The laboratory hoods, although they are meant to provide a sterile environment for designated experiments, could serve as potential site for bacterial contaminations when sterility is compromised.Several species of microorganisms have been isolated across indoor environments in previous studies22. Studies of indoor air qualities and antibiotic susceptibility patterns of bacterial isolates present in most public institutes in developing countries have not been reported so far. This study was designed to characterize the bacterial composition and antibiotic susceptibility patterns of isolates recovered from indoor and immediate-outdoor air of a tertiary research institute in Ghana.An increase in bacterial antimicrobial resistance and emergence of new strains associated with academic research environments is a serious public health challenge and has become increasingly important in recent years. In an environment where inter-personal and research activities are so diverse, bacteria resistance to antimicrobials is a possibilityThis study was conducted between January and May 2017. The study involved determination of bacterial loads and antibiotics susceptibility profiles of the air in selected study sites within the indoors and immediate-outdoors (foyers) of a research institute at the Department of Cell and Molecular Biology (BCMB). Sampling was conducted at different times within the day in duplicates for a period of eight weeks between temperature ranges of 20–32°C. Sampling sites included teaching laboratories (33 × 75 feet), classrooms (32 × 45 feet), experimental laboratories (28 × 40 feet and 12 × 22 feet), laboratory biosafety hoods, foyers (35 × 170 feet and 35 × 40), toilets (8 × 12 feet) and the library (36 × 41 feet) .23. Non-sampled closed plates as controls were included alongside the experiment. Nutrient agar , MacConkey agar , Blood agar  and Mannitol salt agar  plates were exposed for 60 minutes during daily active working hours (8am – 5pm) at different sites. The plates and non-sampled plates controls were incubated at 37°C under aerobic conditions for 24–48 hours.All samples were collected using the open plate passive sampling technique and processed under aseptic conditions following standard microbiological methodset al. (2016)24. Microscopy (Gram’s staining) and biochemical reactions were performed25. Standard plate count was performed to determine the bacterial loads across the sampled sites12. The quantitative measurements of bacteria in colony forming unit per m3 was determined using an equation adapted from Samuel Fakedu26:Bacteria isolates were identified using phenotypic microbiological methods described by Aguilera-Arreola-3) = 5(Number of colonies per Petri dish) × 104 / Dish surface (cm2) × Exposure time (minutes)Colony forming unit  guidelinesClinical Laboratory Standards Institute , cefotaxime (30μg), chloramphenicol (30μg), ciprofloxacin (5 μg), gentamicin (10 μg), nalidixic acid (30 μg), nitrofuratoin (200μg), tetracycline (30μg), penicillin (15 μg), flucloxacillin (5 μg), cloxacillin (10 μg), erythromycyin (5 μg), ceftriaxone (30 μg) and cotrimoxazole (25 μg) .SPSS package version 17.0 andGraphad prism version 6 software.P-values less than 0.05 were considered statistically significant.All statistical analyses were performed usingStaphylococcus sp., Gram positive cocci and Gram negative bacilli, , followed by the toilets (n=11), then the classrooms (n=9) and finally from the library (n=7). The lowest number of isolates was from the railings of the stairways (n=2). A significant number of diverse bacteria was identified from the samples collected across the sampled sites   . The bac people .E. coli andS. aureus  with established antimicrobial profiles were included alongside the experiment. The antimicrobial resistance profile and susceptibility patterns showed that 87.7% and 76.7% of the Gram positive cocci were resistant to Beta Lactam and sulfonamides, 57.1%, 72.4% and 100% of Gram positive bacilli were resistant to Beta Lactam, macrolides and sulfonamides. Resistance to Nitrofurans by Gram negative bacilli was 80.4%, while 84.2% and 87.7% ofStaphylococcus species showed resistance to sulfonamides and Beta Lactam respectively. Resistance to flucloxacillin across the isolates was observed; highest withStaphylococcus sp. Susceptibility of the isolates to tetracycline and gentamycin were observed especially with some Gram positive isolates .As expected, the laboratory biosafety hood had a relatively low percentage of bacterial concentration. Isolation of bacteria from the hoods appears inappropriate as the UV light shield and creates sterile conditions. However, the reasons for the presence of bacteria, albeit at low numbers, might be due to improper disinfection practice, dilution factors of the disinfectants used or/and cross-contamination. In the study, the staircase railings had the lowest percentage of bacteria isolates, contrary to studies conducted elsewhereThe resistance of the bacterial isolates to most of the antibiotics tested in this study calls for serious attention. Both Gram positive and Gram negative bacteria had higher rates of resistance to different classes of antibiotics. Most of the antibiotic classes were used as treatment options by clinicians in case of an infection in the study area. This might limit the antibiotic choice for the treatment of infections associated with these bacteria in the study area. Interestingly, gentamicin and tetracycline showed a level of effectiveness especially against some of the Gram positive bacteria, and these antibiotics might be considered as parts of the treatment regimen in the study area.3) as compared to the World Health Organization standard. Thus, students and workers are at low risk of exposure to airborne bacteria. Isolation of bacteria from the laboratory biosafety hood is of great health concern. Although the majority seems opportunistic, they may have pathogenic potentials with significant consequences. The strength of this study is that it unravels the level of bacterial contamination and subsequent antibiotic susceptibility profiles of a typical working and learning research environments. The antibiotic profiles of the bacterial isolates from the study centre have not been conducted before; the data presented only suggest possible exposure to resistant bacteria strains.In conclusion, the various air sampling sites of the institute showed the presence of bacteria, though with low levels of contamination within the range . For the pictures of the sampled-sites, this will be subjected to further consideration.Controls: Sorry I failed to mention that unsampled controls were included. The controls are unopened media plates. They were subjected to the same conditions as the opened plates.Data: We will consider the kind of information that would be provided by the correlation analysis. And this would be adjusted accordingly if it suits the purpose of the study. Title: Characterization of culturable airborne bacteria and antibiotic susceptibility profiles of indoor and immediate-outdoor environments of a research institute It need to be improved as (mention city and country in the title) - "Characterization of culturable airborne bacteria of indoor and immediate-outdoor environments of a research institute, Accra, Ghana".Material methods: Mention detailed of sampling sites including a graphical diagram. Describe basis of number of samples collected from each site. The manuscript is very basic and need significant enrichment.  Mention climatic condition or meteorological factors during the sampling period.Staphylococcus aureus. All the mannitol fermenters on MSA agar plates are not S. aureus as some of the other Staph sps also ferment mannitol. Authors need to identify S. aureus by using coagulase and thermonuclease tests or by PCR. Authors may refer a paper entitled “Prevalence of Methicillin Resistant Staphylococcal Bioaerosols in and around Residential Houses in an Urban Area in Central India”  for identification of airborneS. aureus. Mention formula for quantitative measurement of bacteria (cfu/m3). Improve methodologies for bacterial identification with special emphasis on identification ofResults:E. coli ATCC25922 andS. aureus ATCC25923) as quality control strains.Serious concern is lack of quality control strains antimicrobial susceptibility testing (AST). Authors should revise AST usingIt would be better if authors mention statistical confidence/significance to represent comparative bacterial count in different sites.Authors are also advised to do statistical correlation analyses of outdoor-indoor movements with bacterial counts to represent effect of movements on indoor bacterial prevalence. Minor comments: CFU is “colony forming unit” not “coliform forming unit”. Major comments:Discussion: needs more focus.I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Excellent review and comments.All the suggestions suitable for the purpose of this study have been considered for article improvements.Many thanks.


Psittacidae are very susceptible to chronic stress and behavioral disorders. We report a successful physiological validation of an enzyme immunoassay for the non-invasive monitoring of adrenocortical activity and, thus, stress in the golden conure. In addition, as an incidental finding, we demonstrate a link between increased glucocorticoid levels and behavioral disorders. Our results are of great relevance for conservation projects and endocrine-behavioral studies of captive golden conures, where the stress evaluation is a fundamental part of animal welfare programs.®) and the other group served as controls. This procedure was reversed afterwards. All birds presented self-mutilation approximately 6 h after the ACTH injection. This behavior disappeared after two days. Peak concentrations  of GCMs were found 6 (4–8) h after ACTH administration; in all (but one) animals also a second peak was found 14 (10–20) h post injection. GCM levels returned to the baseline after 24 h. We physiologically validated a cortisone enzyme immunoassays to measure adrenocortical activity in the golden conure. Such non-invasive methods are important for studies, which are related to welfare, reproductive, and conservation programs. In addition, we could demonstrate a link between increased glucocorticoid levels and behavioral disorders.Psittacidae are very susceptible to chronic stress and behavioral disorders. Information regarding the endocrinology of the golden conure is scarce, especially about adrenocortical activity. Endocrine studies using non-invasive methods are useful, because they allow longitudinal analysis with high numbers of samples without causing additional stress and are viable in the psittacidae management. The objective was to physiologically validate an enzyme immunoassay for measuring glucocorticoid metabolites (GCMs) in this species. Serial droppings were collected from 16 animals. First, one subgroup received adrenocorticotropic hormone (ACTH; Synacthen Depót The golden conure is an endemic species from the Brazilian rain forest. It is found in the extreme west of Maranhão but also in the north of Rondonia and the south of the Amazon River. The distribution of the species partially overlaps the Amazon “deforestation arch”. Over the last few years, the species lost at least 35% of its natural habitat  and is cQuantification of plasma hormones provides valuable information about the endocrine conditions of an animal. However, episodic fluctuations may occur, and these concentrations are also subject to daily and annual rhythms. Moreover, blood collection is a stressful event and can cause, on its own, hormonal alterations ,10. MoniThe application of non-invasive methods for hormone monitoring has increased substantially in mammals and birds . StudiesThus, the objective of this study is to physiologically validate an enzyme immunoassay for measuring glucocorticoid metabolites (GCMs) in droppings of this species. In addition, as an incidental finding, we demonstrate a link between increased glucocorticoid levels and behavioral disorders.This study has been authorized by SISBIO—(Sistema de Autorização e Informação em Biodiversidade) of the Chico Mendes Institute for Biodiversity Conservation—ICMBio, under number 344191-1 and approved by the Ethics Committee of the School of Veterinary Medicine and Animal Science, University of São Paulo, under protocol number 2823/2012 (28/11/2012).Guaruba guarouba) were used . They belonged to the Scientific Breeder Lymington Foundation, located in Juquitiba/SP, Brazil. The birds were kept in stainless steel cages of 1 m height and 1 m2 area, without a nest box and divided in half in order to allocate one individual of each sex in each part. The aviaries had a daily cleaning/disinfection management and food/water changed twice a day (morning and twilight). All birds had water ad libitum. The food provided in the morning was composed of commercial extruded feed , small germinated sunflower seed, varied fruits, vegetables and greens and a slice of green corn cob (2 cm) for each bird three to four times a week. Once a week, boiled egg, wheat germ and vitamin/protein complex  were added. In the afternoon, all birds received the same extruded commercial feed, mix of Psittacidae seed  and varied fruits. For this study, 16 adult and clinically healthy golden conures  into the pectoral muscle and Subgroup 2 was left untreated (controls). After 15 days, Subgroup 2 was ACTH injected (see above) and the other group served as control (untreated). Animals of the two sub-groups did not have visual or auditory contact with the other group during the experiments.The group of golden conures was divided into two subgroups: Subgroup 1  and Subgroup 2 . First, Subgroup 1 received an intramuscular injection of 0.5 mg/kg )p < 0.05). If there was no normality and homogeneity of the variances, the data were transformed (base 10 or X square root logarithm) and, if the transformation was not enough, non-parametrical analysis were performed, such as the Kruskal-Wallis (non-parametric ANOVA) followed by the Dunn (p < 0.05) or Mann Whitney (p < 0.05) tests. All statistical analyses were performed using the program GraphPad InStat 3.01, 32 bit for Windows® 95 version. The obtained data were tested for distribution normality and homogeneity of variances through the Kolmogorov-Smirnov test and the Bartlett test. If the data were considered normal and homogeneous, the One Way ANOVA test was used, followed by the Tukey post-test (®). When we first observed these changes, we started the monitoring with instantaneous sampling (one/zero) every hour until they ended. The normalization of polyuria and the end of the feather pullout was observed 15–20 h and two days after the injection, respectively. Three birds presented blackened stools during a period of 6 to 20 h after administration. All 16 birds pulled out their feathers and presented polyuria about 6 h after the ACTH administration  nor after the ACTH challenges. Thus, the GCM levels of all animals are presented together. Baseline GCM concentrations were 495 ± 106 ng/g (mean ± SD). Following ACTH injection, GCM concentrations started to increase in the first sample (2 h) and reached peak (on average nine times higher than baseline) concentrations 6 (4–8) h  after the administration . In all Guaruba guarouba), with a high number of birds, to physiologically validate a non-invasive method for the evaluation of the adrenocortical activity by measuring glucocorticoid metabolites in excreta. We performed this study in the golden conure (Psittacus erithacus) with self-mutilation were four times higher than those of birds, which did not present such a behavior. Although not in birds, Pizzutto et al. [Although the behavioral observations were not the objective of this study, the feather pullout was an incidental finding that occurred in all the birds involved in the ACTH challenge. Owen and Lane  found tho et al.  also obso et al. , the golo et al. , the moso et al.  and Masoo et al.  affirmedIn vertebrates, an ACTH administration simulates the natural response of the adrenals to stressful stimuli, causing a rapid raise of circulating GC (cortisol or corticosterone) concentrations within a few minutes . The samA study with parrots  reportedOn the basis of a successful physiological validation in the golden conure, we demonstrated that droppings can be used for the non-invasive monitoring of adrenocortical activity by measuring GCMs with a cortisone EIA. In addition, as an incidental finding, we could demonstrate a link between increased glucocorticoid levels and behavioral disorders. Our results are of great relevance for conservation projects and endocrine-behavioral studies of captive golden conures, where stress evaluation is a fundamental part of animal welfare programs.


The ‘Out of India’ hypothesis is often invoked to explain patterns of distribution among Southeast Asian taxa. According to this hypothesis, Southeast Asian taxa originated in Gondwana, diverged from their Gondwanan relatives when the Indian subcontinent rifted from Gondwana in the Late Jurassic, and colonized Southeast Asia when it collided with Eurasia in the early Cenozoic. A growing body of evidence suggests these events were far more complex than previously understood, however. The first quantitative reconstruction of the biogeography of Asian forest scorpions  is presented here. Divergence time estimation, ancestral range estimation, and diversification analyses are used to determine the origins, dispersal and diversification patterns of these scorpions, providing a timeline for their biogeographical history that can be summarized into four major events. (1) Heterometrinae diverged from other Scorpionidae on the African continent after the Indian subcontinent became separated in the Cretaceous. (2) Environmental stresses during the Cretaceous–Tertiary (KT) mass extinction caused range contraction, restricting one clade of Heterometrinae to refugia in southern India (the Western Ghats) and Sri Lanka . (3) Heterometrinae dispersed to Southeast Asia three times during India’s collision with Eurasia, the first dispersal event occurring as the Indian subcontinent brushed up against the western side of Sumatra, and the other two events occurring as India moved closer to Eurasia. (4) Indian Heterometrinae, confined to southern India and Sri Lanka during the KT mass extinction, recolonized the Deccan Plateau and northern India, diversifying into new, more arid habitats after environmental conditions stabilized. These hypotheses, which are congruent with the geological literature and biogeographical analyses of other taxa from South and Southeast Asia, contribute to an improved understanding of the dispersal and diversification patterns of taxa in this biodiverse and geologically complex region. According to this hypothesis, the ancestors of Southeast Asian taxa originated in Gondwana, diverged from their Gondwanan relatives when the Indian subcontinent  rifted from Gondwana, rafted on the subcontinent, and arrived at Southeast Asia during or after it collided with Eurasia in the early Cenozoic. Support for the ‘Out of India’ hypothesis crosses taxonomic boundaries. Research on plants12, fish15, amphibians20, birds21, lizards22, and arthropods23 suggest an Indian origin for many Southeast Asian taxa. The Indian-Eurasian collision also resulted in colonization of the Indian subcontinent by Eurasian taxa, the so-called ‘Into India’ hypothesis, although there are fewer supporting examples25.The ‘Out of India’ hypothesis is often invoked to explain patterns of distribution among Southeast Asian taxa42. Many geologists and biogeographers favor an early collision date, occurring 55–50 Ma at the Paleocene–Eocene boundary42, based largely on geological data including seafloor spreading, tectonic plate movement rates, biostratigraphy, and paleontological data42. This ‘traditional’ hypothesis fails to explain why effects of the Indian-Eurasian collision do not appear until 30 Ma, 20 million years after the collision commenced26.The path of the Indian subcontinent on its northward drift, and the timing of its collision with Eurasia have been extensively debated38. For example, Li et al.19 proposed that rhacophorid frogs first colonized Southeast Asia from India during the Eocene (57–46 Ma). Faunal exchange between India and Southeast Asia ceased during the Middle Eocene but resumed between the Oligocene and Middle Miocene (ca. 34–12 Ma). Although several studies suggest faunal exchange between India and Southeast Asia may have occurred more than once, opinions differ as to how the initial contact occurred. According to Aitchison et al.26 and Ali and Aitchison27, Greater India brushed up against Sumatra and collided with an intra-oceanic island arc, the Dazhuqu Arc, ca. 55 Ma in the Neo-Tethys Ocean. India then moved closer to Eurasia until its final collision, roughly 34 Ma27. Two alternative hypotheses were proposed for the location of India between 55–35 Ma, according to which India was situated either very close to Southeast Asia28 or slightly further away29. The hypothesis of Ali and Aitchison27 was supported by a biogeographical analysis of freshwater crabs, which suggested that dispersal from India to Southeast Asia occurred during the Middle Eocene as India brushed up against Southeast Asia before the final collision9. In contrast, Van Hinsbergen et al.38 proposed that initial contact between India and Eurasia occurred when a microcontinent, the Tibetan Himalayan landmass, rifted from northern India and collided with Eurasia approximately 50 Ma. The rest of the Indian subcontinent was isolated following this initial rift until colliding much later (25–20 Ma). Aitchison and Ali35 refuted the hypothesis of Van Hinsbergen et al.38, suggesting the geological data were misinterpreted.More recent hypotheses account for this discrepancy by suggesting that India experienced several periods of contact with Eurasia, the initial contact occurring as early as 55 Ma, and the final India-Eurasian collision occurring between 35–20 Ma55. These volcanic eruptions, combined with effects of the Chicxulub impact, resulted in drastic environmental changes across the globe and are thought to have caused the Cretaceous–Tertiary (KT) mass extinction55. The Western Ghats of India and the Central Highlands of Sri Lanka have been posited as potential refugia during this period of Deccan volcanism58. Bossuyt et al.17 suggested frog lineages survived in southern India and Sri Lanka during the intense volcanism and subsequently dispersed across Eurasia after India’s collision. Numerous studies identified close relationships between taxa inhabiting the Western Ghats, southern India, and Sri Lanka63. Conti et al.2 proposed that the angiosperm family Crypteroniaceae de Candolle, 1868 reached Southeast Asia from the Indian subcontinent and explained its absence from India as the result of extinction caused by climatic changes  at the end of the Cretaceous and in the early Tertiary but survived in refugia, including Sri Lanka.The hypothesis of Gondwanan taxa rafting on the Indian subcontinent is more complex than the number of colonizations of Southeast Asia, however. Approximately 65 Ma, the Deccan Traps, situated in the center of the subcontinent, underwent extreme volcanic activity, coinciding with the meteorite collision in the Chicxulub Crater, Mexico, and lasting less than a million years64. Asian members of the family, i.e., Heterometrinae Simon, 1879, also known as Asian forest scorpions , the Middle East, the Indian subcontinent, and Southeast Asia73. According to Couzijn23, the ancestor of Heterometrinae and Pandininae Thorell, 1876 originated in eastern Gondwana during the Triassic  is presented here. Divergence time estimation, ancestral range estimation, and diversification analyses are used to determine the origins, dispersal and diversification patterns of the subfamily, and place that in the context of modern understanding of the biogeographical history of South and Southeast Asia.The divergence-time estimation analysis in BEAST produced a fully dichotomous phylogeny. Posterior probability values were generally high and the monophyly of most genera well supported  had the highest evolutionary distinctiveness (ED) among the species ; 0.016 for high extinction rate (ε = 0.9) and low number of missing species (n = 5); 0.040 for low extinction rate (ε = 0) and high number of missing species (n = 50); 0.031 for low extinction rate (ε = 0) and low number of missing species (n = 5). Comparison of AIC values for seven models identified the two-rate Yule model as the best fit for the dataset (Table p = 0.019) suggesting most branching occurred early in the phylogeny. A Monte Carlo Constant Rates (MCCR) test indicated that the gamma statistic was affected by incomplete sampling when at least 28% of the taxa were missing (p > 0.05) (Table Net diversification rates were as follows: 0.023 for high extinction rate (ε = 0.9) and high number of missing species n = 0; 0.016 64 in supporting the monophyly of Asian Heterometrinae and their sister group relationship with African Pandininae, represented in the analysis by Pandinus. This relationship implies Scorpionidae originated on the African continent and Heterometrinae diverged from their African relatives following the separation of West Gondwana (comprising Africa and South America) from East Gondwana . The East–West Gondwana rift has been invoked to explain biogeographical patterns among many taxa76 but the timing of the complete isolation of the Indian subcontinent from Africa has been debated. Prior to the East–West Gondwana rift, India was connected to Africa via Madagascar and Antarctica77, and it is likely Scorpionidae spread across this area. Land connections between India and Africa began to sever ca. 175 Ma78. Rifting began in the north, by opening of the Somali Basin between India-Madagascar and present-day Somalia79, and shortly thereafter in the south, between Antarctica and present-day Mozambique, by seafloor spreading in the Mozambique Basin and opening of the Weddell Sea80. Antarctica was completely separated from West Gondwana by 135 Ma81, and seafloor spreading between Madagascar and Africa had ceased ca. 120 Ma83, ending the rift between West and East Gondwana. However, despite India being physically separated from West Gondwana by this time, mounting evidence suggests India’s biota was not completely isolated from West Gondwana but rather that connections between India and Africa continued well into the Late Cretaceous, possibly even into the early Paleocene89. For example, several Late Cretaceous–early Paleocene fossil tetrapods, discovered in India, reveal close affinities with African relatives, suggesting connectivity between the two landmasses until ca. 65 Ma88. A divergence time estimation of two extant frog clades, based on DNA and calibrated using fossils, also supports a link between India and Africa until roughly 85 Ma in the Late Cretaceous86.The phylogenetic analyses presented here confirm previous studies84 proposed a land bridge, connecting northwestern India to eastern Arabia, known as ‘Greater Somalia’. However, this hypothesis was refuted by geological data90, which demonstrated that, by the Late Triassic–Early Jurassic, Greater Somalia had already broken into two plates, situated far from the Indian subcontinent. Instead, an alternative explanation was proposed, the ‘Oman-Kohistan-Ladakh Island Arc’ hypothesis, which states that an island chain connected the Indian subcontinent with northern Africa between 80 and 65 Ma, or possibly even later93. A major revision of the plate tectonic and paleogeographical model of the Indian subcontinent27 criticized many plate tectonic models of India proposed by biogeographers for failing to consider geological evidence. Ali and Aitchison27 nevertheless supported the notion that the Indian subcontinent was not totally isolated after the East–West Gondwana rift. Their model suggested biotic exchange could have occurred between India and Antarctica-Australia via the Kerguelen Plateau until 90 Ma, and between India and Africa until 85 Ma with a more limited connection via the Madagascar-Providence Bank-Amirante Ridge-Seychelles block27. According to the model, India had reached its maximum isolation by 68 Ma, making it unlikely that faunal exchange would have occurred during this time, although limited connections to Africa may have existed.Several geological explanations have been proposed to explain connections between the faunas of Africa and India in the Late Cretaceous. Chatterjee and Scotese1. Paleogeographical reconstructions of India for this period27 indicate the landmass comprising India and Madagascar drifting northwards along the eastern side of southeastern Africa  and 6800–17,000 Gt of sulphur dioxide (SO2) entering the atmosphere during the most intense phase of volcanic eruption51. Despite the severity of this volcanic activity, the role of the Deccan eruptions in the KT mass extinction has been debated. Indian paleontological records reveal that many terrestrial organisms survived the eruptions50. Consequently, the meteorite impact at the end of the Late Cretaceous, which created the Chicxulub Crater off the Caribbean coast of Mexico, is favored as the primary cause for mass extinctions across the globe, and many studies argue that the effects of Deccan volcanism were only accessory to the environmental changes caused by the meteorite impact53. Some paleontological and geological data suggest the effects of Deccan volcanism were not insignificant, however, leading several researchers to argue that the Deccan eruptions were the primary cause of the global KT mass extinction55. For example, Schoene et al.54 used uranium-lead geochronology to demonstrate that the environmental conditions, which led to mass extinction, initiated during the main phase of eruptions, prior to the Chicxulub meteorite impact, suggesting volcanism was a major contributor to extinction. Palynological data from the Deccan region also indicate a shift in composition of the floral community after these eruptions. The flora was more diverse, and included an abundance of gymnosperms, before the eruptions, whereas angiosperms and pteridophytes dominated afterwards50. Examination of exposed marine beds on India demonstrate a major extinction of planktonic foraminifera with nearly all species disappearing after the last megaflow due to ocean acidification caused by volcanic gases52.At the end of the Cretaceous, starting 67.4 Ma, the Deccan Traps erupted, and India experienced a period of intense volcanism that occurred in three phases, interrupted by nonvolcanic phases, over a short timespan lasting less than 1 million years57. Consequently, these areas have been posited as potential refugia during the KT mass extinction. The results presented here support the hypothesis that the Western Ghats and Central Highlands of Sri Lanka were refugia and suggest Asian scorpionids experienced range contraction during the KT mass extinctions due to stresses caused by the Deccan eruptions, the Chicxulub meteorite impact, or both. The phylogeny and ancestral range estimation analyses support this hypothesis as the ancestor of the largely Indian clade, comprising Chersonesometrus, Deccanometrus, Gigantometrus, Javanimetrus, Sahyadrimetrus and Srilankametrus, was confined to the Western Ghats and Sri Lanka during this period. Sahyadrimetrus and Srilankametrus, endemic to the Western Ghats and Sri Lanka, are basal in the Indian clade, whereas Chersonesometrus, Deccanometrus and Gigantometrus, more widespread on the Indian subcontinent, are more distal. No new lineages of Heterometrinae arose between 92 and 56 Ma  and Deccanometrus xanthopus , which inhabit the savanna and scrub of Pakistan and India, respectively, exhibit reddish-brown coloration, robust pedipalps, metasoma and telson, and short, pale legs unlike the closely related species, Deccanometrus bengalensis  and Deccanometrus phipsoni , which inhabit forests and exhibit uniformly blackish or dark brown coloration, more slender pedipalps, metasoma and telson, and longer legs102. According to the LTT plot  (Chersonesometrus + Heterometrus))), is contradicted by the phylogenetic reconstruction of Heterometrinae presented here and by Loria and Prendini102, i.e., (Heterometrus (Srilankametrus (Javanimetrus (Sahyadrimetrus (Deccanometrus (Chersonesometrus + Gigantometrus)))))).The analyses presented here, as well as evidence from other phylogenetic and biogeographical studies, refute Couzijn’s96 used the Africa-India rift to explain the origins of an Indian clade of Hormuridae Laurie, 1896, the ‘Eurogondwana’ hypothesis104 was invoked to account for the distribution of an Indo-Pacific hormurid clade. According to this hypothesis, Hormuridae originated in Gondwana during the Paleozoic and the ancestors of this Indo-Pacific hormurid clade diverged in isolation from the ancestors of the African and South American hormurid genera, when the Apulian microplate rifted from Africa in the Early Cretaceous105. The Indo-Pacific hormurid clade subsequently arrived in Eurasia when the Apulian microplate collided into Europe and dispersed across the Palearctic region, eventually reaching Southeast Asia and Australasia. No hormurids presently occur in the Palearctic, hence Monod and Prendini96 suggested that the ‘icehouse’ climate in the Neogene and the ‘hothouse’ climate in the late Paleocene–early Eocene may have led to the extinction of these mostly tropical or subtropical, humidity-dependent scorpions.Alternative hypotheses have been proposed to explain the origins and distribution patterns of other Gondwanan scorpions. Although Monod and PrendiniScorpio L., 1758 in northern Africa, the Arabian Peninsula and the Middle East (including part of Iran), suggesting scorpionids were able to survive severe climatic changes such as global warming and cooling. On the other hand, the ‘Out of India’ hypothesis is consistent with the geographical distribution of Pandininae, the sister group of Heterometrinae, which extends from Senegambia across western and central Africa to the Red Sea and the Gulf of Aden, and southward as far as the Zambezi River95. As discussed above, evidence exists that biotic exchange was still occurring between central Africa and India well into the Late Cretaceous and early Paleocene.The Eurogondwana hypothesis is a less likely explanation for the distribution patterns of Asian scorpionids. It implies the absence of scorpionids from the Palearctic, which is contradicted by the presence of 106 produced a chronogram for scorpions, using crown and stem group ages for five arachnid taxa, including the scorpion family Buthidae C.L. Koch, 1837, estimated from fossil data, according to which superfamily Scorpionoidea began to diversify at the end of the Cretaceous, roughly 90 Ma. A biogeographical explanation for the early diversification of Scorpionoidea was not offered. Using five molecular markers and phylogenomic dating, Santibáñez et al.107 recovered the divergence between Heterometrus and Pandinus at the end of the Jurassic (145 Ma), but later108 suggested this divergence occurred in the Late Cenozoic (ca. 20 Ma). Whereas a Jurassic divergence is plausible, based on the present study and the geological literature, a Cenozoic divergence between Heterometrus and Pandinus is unlikely.Sharma et al.109 proposed a Cimmerian continent origin for the Southeast Asian harvestman family Stylocellidae Hansen and Sørensen, 1904. This scenario seems less plausible for Heterometrinae given that Cimmeria rifted from Gondwana ca. 290 Ma, and the dated tree presented here suggests that Heterometrinae diverged from the African Pandininae ca. 115 Ma.ClouseUnderstanding the biogeographical patterns of South and Southeast Asian taxa is confounded by the complex geological history of the region. Despite decades of research, there remains little consensus regarding how and when the Indian subcontinent collided with Eurasia or the events that unfolded during its northward journey. The analyses presented here provide a plausible reconstruction of the origins, dispersal and diversification patterns of another group of taxa, the Asian forest scorpions (Heterometrinae), which can be summarized into four major events. (1) Heterometrinae diverged from other Scorpionidae on the African continent after the Indian subcontinent became separated in the Cretaceous. (2) Environmental stresses during the KT mass extinction resulted in range contraction, restricting Heterometrinae to refugia in southern India (the Western Ghats) and Sri Lanka . (3) Heterometrinae dispersed to Southeast Asia three times during India’s collision with Eurasia, the first dispersal event occurring as the Indian subcontinent brushed up against the western side of Sumatra, and the other two events occurring as India moved closer to Eurasia. (4) Indian Heterometrinae, confined to southern India and Sri Lanka during the KT mass extinction, recolonized the Deccan Plateau and northern India, diversifying into new, more arid habitats after environmental conditions stabilized.102. The dataset was reduced to one terminal per species for the biogeographical analyses presented here, resulting in 35 terminals, representing all seven genera and 31 (76%) species of Heterometrinae, and an outgroup comprising exemplar species of three genera, representing each of the other subfamilies of Scorpionidae, from Africa and the Middle East , representing Pandininae; Scorpio fuscus , representing Scorpioninae Latreille, 1802; and Opistophthalmus capensis , representing Opistophthalminae Rossi, 2016. The tree was rooted on Nebo hierichonticus , representing Nebinae Kraepelin, 1905, the basal clade of Diplocentridae Karsch, 1880, the putative sister group of Scorpionidae64.An extensive reconstruction of the phylogeny of Asian forest scorpions, based on 186 morphological characters and ca. 4200 DNA nucleotides from three mitochondrial and two nuclear gene loci for 132 terminals  is published elsewheredle East : Pandinuc Oxidase Subunit I (COI), 16S rDNA (16S) and 12S rDNA (12S), and two nuclear gene loci, 18S rDNA (18S) and 28S rDNA (28S), were sequenced. Previous research demonstrated this combination of loci was informative for resolving relationships at deep to shallow levels in scorpion phylogeny113. The COI and 18S loci were sequenced in two and three fragments, respectively, whereas all other loci were sequenced in single fragments102. Each locus was sequenced in both the forward and reverse directions. Sequence lengths were calculated using the gc_calculator.py script in Biopython114.Muscle tissue was dissected from the legs of specimens preserved in 95% ethanol, and DNA isolated using the DNEasy Blood and Tissue Extraction Kit . Three mitochondrial gene loci, Cytochrome 116 by auto-aligning with the ‘leavegappyregion’ option selected. The L-INS-I method, an iterative refinement method which uses a local pairwise alignment with an affine gap cost, was selected as the best alignment for all loci117. The aligned sequences were 1761 base-pairs (bp) in length for 18S, 515 bp for 28S, 341 bp for 12S, 493 bp for 16S, and 1078 bp for COI, summing to a total of 4188 bp for the concatenated dataset . The dataset was partitioned by loci with molecular clocks and substitution models unlinked across all partitions. An uncorrelated relaxed molecular clock was applied for each partition and models for individual loci selected using the Bayesian Information Criterion (BIC) in JModeltest v. 2120 on the CIPRES Science Gateway  Africa; (B) Western Ghats and Sri Lanka; (C) Greater Indian Subcontinent; (D) Indochina; (E) Sundaland; (F) Philippines . Areas allowed and areas adjacent matrices  were included as well as a dispersal multiplier matrix with a probability of 1 for adjacent areas, 0.5 for areas with intermediate connections, and 1e−07 for areas with unlikely connection due to separation by large geographical distance or an ocean barrier (Supplementary Table 2). The models Dispersal-Extinction-Cladogenesis, DIVALIKE, and BAYAREALIKE were compared, and an additional parameter (j), which allows a founder speciation event, tested for each (Table 139 phylogenetic diversity (PD) index was applied to all ingroup areas (Table 140 with the R package picante141. The evolutionary distinctiveness (ED) for each species was also calculated using the ‘fair proportions’142 and ‘equal splits’143 methods in picante.Ancestral range reconstruction for the time-calibrated phylogenetic tree was implemented using the BioGeoBEARS v. 1.1.1 package144. The Akaike Information Criterion (AIC) was calculated to determine the best fitting model for the dataset. The command fitdAICrc was applied for the pure birth, birth–death, exponential density-dependent (DDX), linear density-dependent (DDL) and two-rate Yule models (Table fitSPVAR and fitEXVAR were applied for the variable speciation (SPVAR) and variable extinction (EXVAR) models145. Net diversification rates were calculated for a low extinction rate (ε = 0) and a high extinction rate (ε = 0.9), assuming low (n = 5) and high (n = 50) numbers of missing species, respectively. The gamma statistic, which assumes complete sampling, was calculated to determine whether the diversification rate is constant over time, using the gamStat (γ)146 function in the R package ape147. A Monte Carlo constant rates test, conducted using the function mccrTest, was also used to assess whether the observed gamma rate was affected by incomplete sampling, assuming ten different clade sizes  model for comparison. Speciation rates, extinction rates, net diversification rates, and number of rate shifts were calculated by running Bayesian Analysis of Macroevolutionary Mixtures (BAMM) v. 2.5.0149 with the settings nchains = 4, ngen = 10,000,000 and initial priors selected using BAMMtools v. 2.1.6150 in R. The statistical significance of rate shifts was calculated using Bayes factors151. Results were visualized using BAMMtools.Outgroups were removed from the divergence time tree and the tree forced to be ultrametric as described above. A series of analyses were performed on the ingroup tree, to understand diversification patterns within Heterometrinae, using the R package laser v. 2.4-1ls Table . The comSupplementary Information 1.


Cities are the fastest developing ecosystems on the planet. The rapid expansion of urban areas is typically seen as a threat to global biodiversity, yet the role of cities in protecting species that may be rare in the wild remains poorly explored. Here, we report the use of environmental DNA (eDNA) to document the species present in one of the largest urban green spaces in Johannesburg, South Africa. We document a surprisingly large number of taxonomic groups, including some rare and threatened species. Our results support the notion that urban green spaces can provide refuge to a large number of species, and the species inventory provides critical information that can be used by city parks managers to conserve green spaces.Adaptation to environments that are changing as a result of human activities is critical to species’ survival. A large number of species are adapting to, and even thriving in, urban green spaces, but this diversity remains largely undocumented. In the current study, we explored the potential of environmental DNA (eDNA) to document species diversity in one of the largest green spaces in Johannesburg, South Africa. Using a novel metabarcoding approach that assembles short DNA fragments suitable for massively parallel sequencing platforms to the approximate standard ~710 bp COI barcoding fragment, we document the presence of 26 phyla, 52 classes, 134 orders, 289 families, 380 genera and 522 known species from the study site. Our results highlight the critical role that urban areas play in protecting the world’s declining biodiversity. Urban areas are the fastest growing ecosystems on the planet. As cities expand, the surrounding natural habitat is transformed, and wild species are either displaced or are forced to persist in shrinking pockets of fragmented habitats inside cities ,2,3. As Johannesburg is the largest and most populated metropolitan area in South Africa . FoundedTraditional methods of monitoring biodiversity rely on visual observation and morphological identification of species that live in an area ,18. ThesRecent developments in DNA sequencing technologies and high-performance computing have made it possible to study ecological diversity with unprecedented levels of accuracy. Particularly suitable for monitoring the diversity and abundance of species is the massively parallel sequencing of environmental DNA (eDNA) , which iMost DNA-based methodologies are sensitive to the selection of appropriate DNA markers. Elbrecht and Leese (2015)  showed tThe approximate 710 bp length of the COI marker of Folmer et al. (1994) , which iThe current study explores the potential of randomly shearing COI sequences into short fragments, followed by bioinformatic assembly of the complete length of the targeted product of Folmer’s primers. The aims of this study are two-fold. First, it constitutes the first DNA-based survey of species diversity in a South African urban green space, five decades after an ecological restoration project started. Second, we present a cost-effective method for monitoring biodiversity, which uses short shotgun fragments from commonly used sequencing platforms combined with high-throughput bioinformatic pipelines, to approximately the complete ~710 bp length of the COI barcode. The same methodology can be applied to reconstruct the full length of any other DNA fragments.Delta Park is located along the middle reaches of the Braamfontein Spruit (river), a tributary of the Jukskei River which falls within the larger Crocodile (West) and Marico Water Management Area. The upper reach of the river flows through urban areas in the west of Johannesburg while the middle reach of the Braamfontein Spruit is surrounded by formal residential areas. The two dams in the park (Top Dam and Middle Dam) are connected by a narrow channel whose water flows in a north-easterly direction through the park before it joins the Braamfontein Spruit see .Water and sediment samples were collected from three sites within Delta Park: Middle Dam , Top Dam  and the Braamfontein Spruit  . At each® Membrane . Five filters were used for the Middle Dam site, two filters were used for Top Dam, and three filters were used for the Braamfontein Spruit. We changed filters when they became clogged, and DNA was extracted from all the filters. For the sediment, each falcon tube was shaken vigorously and approximately 5 mg of the homogenized slurry was subsampled for DNA extraction.Environmental samples were processed in a room that has not been used for any DNA work prior to this study. All surfaces were wiped down with 100% bleach, left to dry, and subsequently wiped with 70% EtOH. Approximately 500 mL of the collected water was filtered using a MicroFunnelTM Filter Unit with 0.2 µm Supor® III EDTA, 20 mM Na-Bisulfit and 4% polyvinylpyrrolidone 40) using a Covaris sonicator . Ground specimens were digested using 200 µL of lysis buffer , 10% SDS and 20 µL Proteinase K in a 1.5 µL Eppendorf tube. The samples were left to incubate for one hour at 60 °C. Lysates were centrifuged (4600 RPM) for 10 min at 4 °C. The supernatant was transferred into a new 1.5 µL Eppendorf tube, and 250 µL buffer mix III (3 M KAc) was added to each tube. The tubes were left to incubate on ice for 10 min followed by a 15 min centrifugation step at 4 °C. The supernatant was transferred onto a glass fibre filter plate and was centrifuged for an additional 5 min at 4 °C. Approximately 280 µL of isopropanol was added to the filtrate, which was then vortexed vigorously and left to incubate on the plate for 20 min at −20 °C. Each plate was centrifuged for an additional 25 min at 4 °C. Finally, 200 µL of ice cold 70% EtOH was added to each tube, which was centrifuged for 5 min at 4 °C. The resulting DNA pellets were dried at room temperature and then dissolved in 50 µL TE buffer.Metagenomic DNA was extracted by grinding sediment and water filters in 200 µL grinding buffer . Thermal cycles for each reaction started with an initial denaturation step at 95 °C for 15 min, followed by 25 cycles of denaturing steps at 95 °C for 20 s, an annealing stage of 49 °C for 45 s, and an elongation step at 72 °C for 1 min. This was followed by a final elongation step at 72 °C for 10 min. To minimize the amplification of non-specific PCR product, we selected 25 PCR cycles, as preliminary analyses indicated that this produced sufficient product for Illumina library preparations.A portion of the mitochondrial COI gene was amplified using 3 µL of template DNA, 6 µM of universal primers LCO1490  and HCO2198  , 1× PCR Equimolar concentrations of amplicons from sediment and water samples were pooled into two separate tubes and randomly sheared into approximately 250–300 bp fragments using a Covaris sonicator . To maximize the random shearing of the COI amplicons, multiple rounds of sonication with different intensities were performed, and the size of the resulting fragments after each round of sonication was checked using a Bioanalyzer 2100 . Only fragments within the expected range were selected for genomic DNA library preparation. The targeted sequence was treated as a miniature genome that was sheared to a large number of smaller overlapping fragments that is necessary to assemble longer fragments.® UltraTM DNA Library Prep Kit  and sequenced on an Illumina HiSeq 4000 platform  using 2 × 150 bp paired-end chemistry according to the manufacturer’s instructions.Genomic libraries were generated using a NEBNextLow-quality sequences and adapter contaminants were identified in FastQC  and remoThe assembled sequences were dereplicated into unique sequence features using VSEARCH v2.4.2 , chimerihttps://www.ncbi.nlm.nih.gov/refseq/), using the MEGABLAST package (which searches for ‘highly similar’ matches) [−5. The four best matches for each query were retained and reported. A Last Common Ancestor (LCA) consensus taxonomic rank was assigned to each sequence in BASTA v1.3.2.3 (https://github.com/timkahlke/BASTA) [All consensus sequences were searched against an in-house database of COI sequences extracted from the NCBI non-redundant nucleotide database (matches)  and somee/BASTA) . For eace/BASTA)  and visue/BASTA) .To assess the efficiency of the sequence assembly and taxonomic rank assignments, all sequences in our dataset were divided into two approximately equally sized fragments using FASTX-Toolkit v 0.0.8 . Small oAlpha diversity indices, Shannon , SimpsonThe Illumina sequencing run yielded 5,028,734 and 5,219,475 paired-end raw sequences from water and sediment samples, respectively. MEGAHIT assembled quality-filtered sequences from water samples into 2208 and 5443 contigs, respectively. The cumulative length distribution histogram of raw assemblies shows that the length of less than 5 percent of the sequences exceeded 712 bp .Achlya bisexualis, Aspergillus tubingensis, Biomphalaria glabrata, Bulinus natalensis, Cheyletus malaccensis, Chrysomya rufifacies, Drosophila hydei, Fannia canicularis, Homo sapiens, Opistophthalmus boehmi, Rattus norvegicus, and Tuberolachnus salignus, are known to occur in Delta Park based on earlier, non-genetic studies , into 26 phyla, 52 classes, 134 orders, 289 families, 380 genera, and 522 known species. Among these, only 12 species, namely  studies . More thThe exact NCBI accession number of the best matches for the first half of the assembled sequences were matched among four best matches of the second half in more than 98.4% of the pair-wise comparisons. In almost all cases, the exact match for NCBI accession number of the first half was found among the top eight blast hits for the second half. The BASTA pipeline assigns a taxonomic rank of “unknown” or “unknown-eukaryotic” to all manually generated chimeric sequences. All these confirm that the negative effects of chimeric sequences in our dataset are likely to be minimal.Shannon’s diversity index was estimated slightly higher for sediment compared to water samples (sediment H = 2.13 and water H = 2.025), while both the Evenness index and the Simpson’s index were higher for the water communities .Cities constitute the newest extensions to wild habitats. The role that urban ecosystems play in preserving the world’s declining biodiversity represents an underappreciated area of research , yet it Afrarchaea, reported from South Africa [Not surprisingly given their abundance, arthropods dominated the aquatic biological diversity of Delta Park. Arthropods were followed by species belonging to the phyla Oomycota, Mollusca, Cnidaria, Chordata, Rotifera and Annelida. High diversity of arthropods has already been reported from urban areas in South Africa  and elseh Africa , their ph Africa ,63,64,65Delta Park is home to several firefly species. Populations of fireflies are declining across the globe as a result of high intensity artificial light at night (ALAN) ,67,68 thRattus sp.), cattle , and a number of unidentified species belonging to Gekkonidae, Scincidae and Eulipotyphla families . Among aquatic vertebrates, species of Perciformes, Blenniidae, Cypriniformes, Clarias sp. and Galaxiidae fish were identified. While some of these species such Clarias sp. are common inhabitants of freshwater ecosystems worldwide and in southern Africa, the presence of some other species most likely represents non-native species released into dams by aquarium owners.Environmental DNA highlighted the presence of several vertebrate species in Delta Park. These include rats . Infection with schistosome flatworms can progress to the development of schistosomiasis, the world’s third most devastating parasitic disease [Acanthamoeba sp., the causative agent for ocular keratitis and granulomatous encephalitis of the central nervous system, is also a major health concern [Delta Park is home to two species of freshwater aquatic snails,  disease . The ide concern .The primers used in the current study were initially developed for animals. However, the identification of the several species of fungi and algae in our dataset points to the potential of the COI marker for the identification of additional taxa, although only at higher taxonomic ranks , becauseWhile we cannot rule out that some of the sequences that have failed conclusive assignment to lower taxonomic ranks are artefacts of the bioinformatic assembly, others undoubtedly reflect the lack of publicly available records for the species in question, or for their close relatives.Methods that are based on the last common ancestor to assign taxonomic rank perform poorly when local databases are not complete, which is a common problem in underrepresented geographic areas such as Africa , as wellAs scientific efforts to characterize global biodiversity using environmental DNA intensify, our study emphasizes that the need for a comprehensive taxonomically-curated reference database is equally important. A reference database to address such shortcomings requires close cooperation between experts from different fields, such as systematics, morphology, biochemistry and molecular biology, at both regional and global scales. Successful collaborations can have far-reaching implications for better characterizing global biodiversity .We demonstrated that environmental DNA obtained from water and sediment samples can be used to detect the presence of species in aquatic habitats, even if a species is present at densities undetected during visual biodiversity surveys. This reaffirms that aquatic habitats retain a molecular imprint of species inhabiting the area . HoweverThe specific methodology applied in the current study, which is based on the sequence assembly, results in different sections of the markers being covered with different number of raw sequences. This heterogeneous coverage across the length of the amplicon limits the power of such methodologies to estimate the abundance of each species. However, presence or absence of species in an area can be reliably investigated.


CCN6 of five patients with PPD from China in order to increase the clinical data of PPD.No formal diagnostic criteria for progressive pseudo‐rheumatoid dysplasia (PPD) are available because of insufficient clinical data, which results in that PPD is often misdiagnosed with other diseases. Whole exome sequencing (WES) and Sanger sequencing were employed to reveal the novel mutations on Four suspected PPD pedigrees containing five patients in total were collected from 1998 to 2018 in our medical center. The phenotypes of each suspected PPD case were recorded in detail, and peripheral blood samples were collected for subsequent sequencing. Genomic DNA was extracted from peripheral blood samples, and Agilent liquid phase chip capture system was utilized for efficient enrichment of whole exome region DNA. After acquiring raw sequenced reads of whole exome region, bioinformatics analysis was completed in conjunction with reference or genome sequence (GRCh37/hg19). Sanger sequencing was performed to identify the results of WES.CCN6 gene were identified including (CCN6):c.643 + 2T>C, (CCN6):c.1064_1065dupGT, (CCN6):c.1064G > A), and exon4:c.670dupA:p.W223fs.In total, four novel PPD‐related mutation sites in CCN6 mutation spectrum, the clinical symptoms and signs. Moreover, the study highlights the utility of WES in reaching definitive diagnoses for PPD.Our findings increase the clinical data of PPD including the  CCN6 gene were first reported. Rare symptoms and signs of PPD patients were recorded and further increased physician's awareness of this disease.Whole exome sequencing is a time‐ and cost‐effective method to identify the gene mutations for some rare diseases including PPD. Four novel mutations on  The estimated prevalence rate of PPD is 1/1,000,000 in the United Kingdom but varies regionally worldwide. The prevalence rates reported in the Middle East and Gulf States, Turkey, India, and other endogamic communities are markedly higher than that of the United Kingdom , a rare autosomal recessive genetic disease  in five affected individuals. In addition, some rare clinical features, such as flexion deformity of elbows, were also reported. Overall, the aim of this study was to highlight some rare clinical features, radiographic features, and novel mutations of PPD to increase the awareness of this disorder among clinicians, thereby avoiding inappropriate treatment (such as antirheumatic treatment) and helping to relieve the associated pain and disability to improve the quality of life of the patient.In this study, the genetic characterization of four multiplex Chinese pedigrees displaying similar uncharacterized skeletal dysplasia was confirmed using WES and subsequent Sanger sequencing. Specifically, we identified four novel mutations in the 22.1The study protocol was approved by the Ethics Committee of Peking Union Medical College Hospital (PUMCH). All the experiments were performed in accordance with relevant guidelines and regulations.2.2Four suspected PPD pedigrees containing five patients in total were collected from 1998 to 2018 in PUMCH. The phenotypes of each suspected PPD case were recorded in detail from the time the patients were admitted to PUMCH. The first symptom of all five patients appeared between the ages of 3 and 8 years, whereas no symptoms were noted in infancy.Pedigree 1, originating from Hunan province of China, comprised one proband and seven other family members across three generations  . Genomic DNA was extracted from peripheral blood samples using a QIA amp DNA Blood Mini Kit (Thermo Fisher) according to the manufacturer's protocol. Agarose gel electrophoresis was performed to analyze the degradation level of DNA and detect possible RNA or protein contamination. Qubit 4 (Thermo Fisher) was used for the precise quantification of extracted DNA.2.4Next‐generation sequencing, especially WES, is of considerable value for the diagnosis of genetic diseases as exon mutations underlie > 85% of all genetic diseases related to DNA mutations . The process mainly consisted of three steps based on Sorting Intolerant From Tolerant (SIFT), PolyPhen2, and Mutation Taster software: step 1, quality evaluation of the sequencing data including analysis of the sequencing error rate along, sequencing depth and coverage, and the comparative rate; step 2, variation testing; step 3, variation screening and disease correlation prediction Figure .2.6To verify the reliability of the WES results, 22 samples, independent from the samples for WES, were obtained from all members in the four pedigrees and applied to Sanger sequencing by the sequencing instrument (Applied Biosystems Inc.). Sequence analysis was performed using Chromas software . To identify mutation sites, sequencing results were compared with reference sequence, along with their parents’ sequences. The genotype was obtained by analyzing the type of bases at the mutation sites.3In total, five patients  were diagnosed clinically with PPD. The average age at onset of PPD was 5.4 years (3–8 years) and at diagnosis was 19 years (16–25 years). Among all enrolled patients with PPD, the diagnosis was supported by clinical features, radiological features, and laboratory findings.The initial symptoms in most enrolled patients with PPD were stiffness and swelling of interphalangeal joints Figure . InteresGarcia Segarra N et al. reported metaphyseal enlargement of the interphalangeal joints as an early radiological finding :c.643 + 2T>C, NM_198239.1 (CCN6):c.1064_1065dupGT, NM_198239.1(CCN6):c.1064G > A, and NM_198239:exon4:c.670dupA:p.W223fs. Notably, the patient in pedigree 4 harbored a novel compound heterozygous mutation in CCN6 (NM_198239.1) including maternal NM_198239.1(CCN6):c.643 + 2T>C and paternal NM_198239.1(CCN6):c.1064_1065dupGTfsTer33 from heterozygous parents. In addition, NM_198239:exon4:c.670dupA:p.W223fs (frameshift mutation) and NM_003880:exon5:c.616dupA:p.W205fs (frameshift mutation) were identified as a novel compound heterozygous mutation of the CCN6 in pedigrees 1 and 3. All the mutations were located in different regions  and/or new mutation sites on the CCN6. In this study, four novel mutation sites on CCN6 were found through WES.Four pedigrees were enrolled in the present study based on the typical clinical and radiological features of five probands who presented with multiarticular involvement, such as swollen metaphysis, deformity, reduced range of motion, and narrow joint space of the interphalangeal joints, wrists, elbows, hips, knees, and ankles. Aside from abnormal gait, the lack of systemic inflammation, as evidenced by normal RF, CCP, ESR, and CRP, also supported the diagnosis of PPD. All the symptoms and signs found in the five probands were consistent with those from published studies  gene family that regulate cell proliferation, cell differentiation, and cell migration in connective tissue :c.643 + 2T>C, NM_198239.1(CCN6):c.1064_1065dupGTfsTer33, NM_198239.1(CCN6):c.1064G > A, and NM_198239:exon4:c.670dupA:p.W223fs. The four novel mutation sites were predicted to be crucial to the onset of PPD. Specifically, the T to C transduction in intronic DNA occurred two bases away from CCN6 cDNA base 643 in the exon–intron boundary, which constitutes sequence necessary for recognition by the splicing machinery. Such splicing mutations are prone to posttranscriptional RNA splicing error, thus leading to protein changes and, for CCN6, the likely onset of PPD. The GT duplication causes the subsequent substitution of glutamine to valine at position 356 in the CCN6 protein sequence with the frameshift altering the subsequent amino acids as well, thereby likely disrupting protein function. Similar protein disruption was considered to result from the G to A transition in exon 5 and the A duplications.In the present study, four novel mutations in the CCN6 elucidated in our study may further enhance our understanding of the correlation between CCN6 protein function and PPD pathogenesis. Our study revealed the genotype of patient with PPD using WES technology in Chinese pedigrees, and our results underscored the utility of WES in arriving at a definitive diagnosis for rare skeletal dysplasia.Through combination of the sequencing results with the complete clinical documentations, five patients in the four pedigrees were diagnosed with PPD, supporting that WES combined with Sanger sequencing can be of considerable assistance in disease screening for families with hereditary disease. The novel mutation sites on the CCN6 increases the expression level of collagen type II, aggrecan, and the transcription factor SOX9, stimulates superoxide dismutase activity (Davis, Chen, & Sen, CCN6 promotes the precursor cell differentiation of human chondrocytes (Wang et al., CCN6 did not lead to any pathologic changes in mice, whereas CCN6 over‐expression downregulated bone morphogenetic protein and Wnt pathways in zebra fish (Hann, Kvenvold, Newby, Hong, & Warman, CCN6 expression in patients with PPD did not appear to markedly influence such pathways. Alternatively, hyperproliferative chondrocytes consequent to low CCN6 expression levels may explain the enlarged metaphysis in patients. In addition, the progressive narrowing of all joint spaces and reduced joint mobility may be due to delayed differentiation of chondrocyte precursor cells, with concomitant reduced collagen type II synthesis and secretion to the extracellular matrix (Bhavani, Shah, Shukla, Dalal, & Girisha, It has been shown in vitro that The clinical features of PPD overlap those of juvenile rheumatoid arthritis, mucopolysaccharidosis, and ankylosing spondylitis. Although the symptoms of PPD may appear in early childhood, patients with PPD are often misdiagnosed or receive a delayed diagnosis until joints and spine deformities become noticeable (Ekbote et al., CCN6 mutations before further screening the genotypes of patients and other members of their family. Overall, the five probands presented similar clinical phenotypes but no specific correlation between genotypes and phenotypes was detected. Nevertheless, the results of the study expanded the CCN6 mutation spectrum that is associated with PPD and may aid in further elucidating the function of CCN6. In the future, it will be necessary to establish and perfect the diagnostic criteria for PPD. Additionally, more effective and cost‐effective gene panel sequencing should be validated in order to make a definite diagnosis of PPD based on typical signs and symptoms combined with the results of gene panel sequencing. Finally, additional attention should be paid to developing effective and efficient treatments for PPD, such as gene therapy and targeted biologics.In conclusion, the findings of our study increased the evidence to support timely diagnosis and treatment of PPD. In particular, the results demonstrated that WES was helpful to ascertain The authors declare that there is no conflict of interest.Yingjie Wang and Ke Xiao were involved in preparation of the research images, review of the literature, and writing the paper. Yuemei Yang and Jin Jin were involved in collection of the data and some data analysis. Zhihong Wu, Qiu Guixing, Xiuli Zhao, and Xisheng Weng were involved in provision of research ideas and review of the paper. All authors have seen and agree with the contents of the manuscript.Written informed consents for publication of this research and accompanying images were obtained from all participants or, if the subjects were under 18 years of age, from a parent or legal guardian.