Saccharomyces cerevisiae) has broad genetic homology to human cells. Although typically grown as 1-2mm diameter colonies under certain conditions yeast can form very large (10 + mm in diameter) or ‘giant’ colonies on agar. Giant yeast colonies have been used to study diverse biomedical processes such as cell survival, aging, and the response to cancer pharmacogenomics. Such colonies evolve dynamically into complex stratified structures that respond differentially to environmental cues. Ammonia production, gravity driven ammonia convection, and shear defense responses are key differentiation signals for cell death and reactive oxygen system pathways in these colonies. The response to these signals can be modulated by experimental interventions such as agar composition, gene deletion and application of pharmaceuticals. In this study we used physical factors including colony rotation and microgravity to modify ammonia convection and shear stress as environmental cues and observed differences in the responses of both ammonia dependent and stress response dependent pathways We found that the effects of random positioning are distinct from rotation. Furthermore, both true and simulated microgravity exacerbated both cellular redox responses and apoptosis. These changes were largely shear-response dependent but each model had a unique response signature as measured by shear stress genes and the promoter set which regulates them These physical techniques permitted a graded manipulation of both convection and ammonia signaling and are primed to substantially contribute to our understanding of the mechanisms of drug action, cell aging, and colony differentiation.Baker’s yeast ( Saccharomyces cerevisiae has broad genomic homology to human cells and is simple to grow and genetically manipulate  and in two ground-based simulations of microgravity: rotation and random positioning. Rotating culture devices have been a popular model to mimic some of the physical factors induced by microgravity during spaceflight . Table Second, we evaluated the dependence of two stress response genes, SSA4 and RPL34b, on the stress promoters, Msn4 and Sfp1 ), was used to match our earlier studies   in the yeast genome as described previously . The PHAB is a sealed container that allows for gas exchange but meets NASA safety requirements for levels of containment. (https://www.nasa.gov/centers/johnson/engineering/life_support_systems/crew_payload/index.html). PHABs were placed in cold bags at 4∘C, and remained chilled for five days during handoff to the SpaceX-8 rocket. Flight samples were flown to the International Space Station (ISS). Approximately two days post-launch, the growth of the yeast on ISS was initiated by transferring the PHABs to Commercial Generic Bioprocessing Apparatus 4 (CGBA4) . After 28 days in flight, CGBA4 was commanded down to + 4 ∘C. The following day the PHABs were moved back to cold bags, and returned to Earth the next day on the Dragon capsule. Plates remained chilled for a total of six days during undocking, return to Earth, salvage, and transport to the lab for flow cytometry analysis.Figure The second set of plates, the static flight controls were loaded into a second set of PHABs at Kennedy Space Center and handled to match the exact timing and temperature profile of the flight samples. Ground controls were conducted with a 24-hour offset relative to the flight samples and the static controls in PHABs followed the same temperature changes as the flown samples.∘C) rather than 30 ∘C and they were not placed in PHABs. To control for the difference, an additional set of static ground controls were maintained in plastic bags at 25∘C and also not placed in PHABs. Results from the two set of static controls, PHABs/30 ∘C versus no-PHAB/25 ∘C, did not differ (Pearson correlation > 95%) so data from the two were pooled in the statistical analyzes. Similarly, we could not detect any differences due to plate position of the replication using any of the modalities studied (Pearson correlation > 95%).A third and fourth set of samples were subjected to Wall-Vessel rotation or random positioning (RPM) during growth. The timing of the rotating and RPM cultures matched that of the flight samples with the exception that they were grown at ambient laboratory conditions  oriented with the surface of the agar perpendicular to the floor see Fig. . Plates Flight, rotation, and RPM growth conditions had four replicates on the parental strains and six replicates for the deletion strains. Static cultures had eight replicates for the flight, rotation, and RPM conditions and 12 replicates for the static cultures.μ l aliquots of yeast in PBS were placed in 5ml tubes preloaded with 300 μ l of propidium iodide (PI) at 1 μ g/ml solution to measure viability, or 300 μ l of dihydroethidium (DHE) at 5 μ g/ml to measure reactive oxygen.All assays were performed by flow cytometry. Our probes for viability, GFP expression, and yeast redox levels have been validated previously , digested for 30 min at 28 ∘C with 5.5% glusuase and 15 Um/ml lyticase in sorbitol buffer, and washed twice in kit binding buffer to which 1.2 M sorbitol is added. Yeast were resuspended in 100 μ l binding buffer with sorbitol and stained with 5 μ l of annexin and 1 μ l of PI at 0.1 mg/ml in binding buffer with sorbitol for 15 min at room temperature for 15 min. 400 μ l binding buffer with sorbitol was added and the samples were maintained on ice until analyzedTo measure caspase yeast were washed once in PBS, pelleted, incubated for 20 min at 28 ∘C for three minutes and by exposing yeast to 120 mM acetic acid for five minutes. Negative controls were generated using liquid yeast cultures in log phase growth.Positive controls for the PI, DHE, annexin binding, and caspase assays were generated by heating yeast to 40 Flow cytometry of PI-stained cells revealed three peaks - high uptake, low uptake, and negative uptake. Cell death was identified as high PI uptake . Flow cytometric studies were carried on a Becton Dickinson  LSRII cell analyzer flow cytometer using Excitation/Emission of 510/580nm, 532/610nm, and 488/530 nm for DHE, PI, and GFP/ CaspACE™ FITC-VAD-FMK/annexin, respectively. The instrument was aligned using fluorescent beads. Yeast cell gating was established using a display of log side scatter fluorescence verses linear forward scatter, using glutaraldehyde stained pan fluorescent controls for confirmation in fluorescent channels. Unstained yeast samples were used to determine levels of background fluorescence Measurements were collected on at least 10,000 cells per sample.Flow cytometry data is presented as geometric mean ±standard error of the mean of fluorescence in arbitrary units compared to a negative control with a minimum of six replicates. Geometric means were determined using FlowJo Software . Analysis of variance and post-hoc comparison using Tukey’s test was performed using Statistica 6.1 , using correlation matrix product moment and partial correlations. Flow cytometry data was also analyzed statistically using Kolgomorov-Smirnov summation statistics Young . SpecifiExpression of the stress response genes, SSA4 and RPL34b, was measured using eGFP fusion protein expression clones. Stress promoter dependence was assessed by comparing the eGFP signal of the parent strains to clones in which the Msn4 or Sfp1 promoter genes had been deleted Fig. .In summary, in colonies grown on rich YPD media, SSA4 increased compared to static culture in all culture modalities. The increases in SSA4 expression seen in flight and rotation, but not random positioning, were Sfp1 dependent. RPL34B gene expression did not change during any culture condition on YPD. Flight and rotation had oppositely directed RPL34B gene expression changes dependent on Msn4.Colonies grown on nutrient poor YE media yielded gene expression changes distinct from those seen in nutrient rich YPD media. SSA4 expression increased in flight and was Sfp1dependent. Ssa4 expression did not change during rotation and decreased during random positioning. In both rotation and random positioning, expression of SSA4 was dependent on both Msn4 and Sfp1. On YE media, RPL34B increased in flight with Msn4 dependence, but did not change during rotation or random positioning.Expression of ATO3, INO1, TRR1, BEM1 and EDC3 is shown in Fig. Cell death by necrosis and apoptosis (as measured by PI and Annexin V staining) was affected by the culture modalities and manifested diverse stress defense promoter dependence Fig. . In summChanges in Annexin V exposure and caspase activity (indicative of apoptosis) were consistent with the changes in the middle peak of propidium iodide stained cells (e.g. low PI uptake) observed by flow cytometry. The specific fluorescent values and statistical parameters for DHE staining are tabulated in Table Reactive oxygen species (ROS) production, especially superoxide formation, was monitored with DHE. Positive control cells were induced with acetic acid and heat (data not shown). On YE, none of the culture techniques changed DHE signal, but basal levels were Sfp1 and Msn4 dependent. The specific fluorescent values and statistical parameters for DHE staining are tabulated in Table Systematic studies of the effects of true microgravity and common microgravity simulations on cell physiology has been lacking. Furthermore, how these environments combine with other fundamental variables such as growth media to influence the cellular response to stress is an outstanding question. We address both of these by examining cellular changes, including gene expression (reported by shear stress promoters Msn4 and Sfp1), reactive oxygen species, and apoptosis.E. coli where nutrient depletion in simulated microgravity induced stress response genes , thioredoxin reductase (TRR1), and stress seventy subfamily A (SSA4). These changes were independent of media type. Interestingly, both INO1 and TRR1 are predominantly expressed in the L or lower colony cells when yeast colony stratify into feeder and growing layers in giant colonies (Cap et al. Cell death by necrosis and apoptosis increased in flight as shown by multiple modalities: uptake of propidium iodide up, expression of caspase, and binding of annexin V. Induction of apoptosis by simulated microgravity has been seen with other cell types including human lymphocytes (Ward et al. The changes in INO1 and SSA4 genes are significant in both random positioning with gravity vector averaging and rotation techniques but oppositely directed to the space flight changes, underscoring the uniqueness of the effects of true microgravity. During culture on the ISS, yeast cultures are also exposed to increased radiation (Nislow et al. We found differences in the responses of yeast in the rotating wall vessel versus random positioning models in both ammonia-dependent and stress-dependent changes, providing clarity to the controversy as to whether the random positioning device is a form of rotation, or a distinct stimulus Klaus . These dThe ability of physical methods to manipulate yeast colony redox, apoptosis, and stress defense status is important at multiple levels of organismal biology. Gene deletion, although targeted to a single locus, will often cause follow-on effects in multiple integrated pathways, especially when promoters are deleted (Park et al.


Neonatal gastrointestinal (GI) bacterial community structure may be related to bacterial communities of the mother, including those of her milk. However, very little is known about the diversity in and relationships among complex bacterial communities in mother-infant dyads.Our primary objective was to assess whether microbiomes of milk are associated with those of oral and fecal samples of healthy lactating women and their infants.16S ribosomal RNA (rRNA) gene. Alpha and beta diversity indices were used to compare microbiomes across time and sample types. Membership and composition of microbiomes were analyzed using nonmetric multidimensional scaling and canonical correlation analysis (CCA). The contribution of various bacterial communities of the mother-infant dyad to both milk and infant fecal bacterial communities were estimated using SourceTracker2.Samples were collected 9 times from day 2 to 6 mo postpartum from 21 healthy lactating women and their infants. Milk was collected via complete breast expression, oral samples via swabs, and fecal samples from tissue (mothers) and diapers (infants). Microbiomes were characterized using high-throughput sequencing of the Streptococcus , whereas Bacteroides were predominant in maternal and infant fecal microbiomes . The milk microbiome was more similar to the infant oral microbiome than the infant fecal microbiome. However, CCA suggested strong associations between the complex microbial communities of milk and those of all other sample types collected.Bacterial community structures were relatively unique to each sample type. The most abundant genus in milk and maternal and infant oral samples was These findings suggest complex microbial interactions between breastfeeding mothers and their infants and support the hypothesis that variation in the milk microbiome may influence the infant GI microbiome. High-throughput sequencing has unveiled distinct and complex consortia of microbial communities in and on the human body e.g., , and theStreptococcus  characterize bacterial diversity in the various sample types over time, 2) assess the dissimilarity/similarity of the bacterial communities among the sample types over time, and 3) assess multivariate relations among bacterial communities of the different sample types. Our overarching a priori hypothesis was that the milk microbiome would be more similar to that of infant feces than the other biological niches investigated. It is noteworthy that the composition of maternal fecal and milk microbiomes from this study have been described previously  were exclusively fed the milk of their mother until they were 3 mo of age. Most other infants (n = 10) were predominantly fed the milk of their mother but occasionally received fluids such as water, glucose-water, and other liquids during the first 3 mo.This study was carried out as a prospective, longitudinal investigation of 21 healthy, breastfeeding women recruited in their third trimester of pregnancy from the Pullman, WA/Moscow, ID, area. Samples and data were collected on 2, 5, and 10 d (±1 d), and 1, 2, 3, 4, 5, and 6 mo (±1 d) postpartum between June 2011 and April 2012. Mothers needed to be ≥18 y old and planning to breastfeed for ≥6 mo to participate. Nonexclusive breastfeeding was not a reason for exclusion, although most infants . Samples collected at a university site were immediately stored at −80°C; those collected at a home or hospital site were kept on ice until they could be frozen at −80°C.g for 10 min at 4°C), the supernatant decanted, and the remaining pellet resuspended in sterile 0.5 mL TE50 . Samples were subjected to enzymatic and physical lysis as described previously  following the manufacturer's protocol.16S ribosomal RNA (rRNA) gene. Descriptions of the primers used have been reported previously  was pooled to create a composite sample with processing and sequencing as described previously . Raw DNAIndividual-based rarefaction and accumulation curves were generated using the rarecurve and specaccum functions, respectively, in the vegan package (v2.4.6) in R , 33. IndP) values were adjusted for multiple comparisons using a Bonferroni correction. Significance for the specified responses was declared at P ≤ 0.05.Alpha diversity, describing “within” sample diversity , 35, wasBeta diversity (“between” sample diversity) was examined using principal coordinates analysis (PCoA) using Hellinger-transformed data and nonmetric dimensional scaling (NMDS) using the Bray–Curtis dissimilarity matrix. PCoA and NMDS are both ordination techniques that visualize the similarity or dissimilarity of various bacterial communities . AnalysiTo further investigate relations among bacterial communities of different sample types, we conducted canonical correlation analyses CCAs;  among pa1) the contribution of maternal oral, milk, and fecal , and also infant oral bacteria to the bacterial composition of the infant fecal microbiome, and 2) the contribution of maternal fecal, oral, and infant oral bacteria to the milk microbiome. For both estimations, rarefied sequence count data were used.Additionally, SourceTracker2  were sequenced yielding a total of 18,534,383 sequencing reads  following demultiplexing and sequence read processing, 1,022 taxa were identified. After removing duplicates and samples with <2,000 reads, 791 samples  were used in the analyses  and the P < 0.005) for each of the diversity indices. As such, we examined the effect of time on diversity indices within each sample type and the effect of sample type across time (P < 0.0001) than infant feces and oral samples. The Shannon diversity of maternal feces was also greater (P < 0.008) than most sample types at each time point.A sample type by time interaction was evident ; whereas the most abundant genus in infant and maternal feces was Bacteroides . The 5 most abundant genera in both milk and infant oral samples were Streptococcus, Staphylococcus, Gemella, Rothia, and Veillonella. With the exception of Staphylococcus, these genera were also the most abundant in maternal oral samples.Average relative abundances of the 4 most abundant phyla are presented in Supplemental Figure 1). Both ANOSIM  and adonis  tests at the genus level indicated an effect of sample type on complex bacterial community membership, supporting the visual clustering observed in the NMDS plots. NMDS plots suggested that milk and infant fecal samples have some similarity in early life but become increasingly different over time. Conversely, milk and infant oral microbiomes appear to become increasingly similar to each other and to the maternal oral microbiome over time.NMDS and PCoA analyses revealed similar results; since the amount of variation explained by the first 2 components of the PCoA at the genus level was only ∼21%, only NMDS results are reported here. There was clustering by sample type despite some overlap . When all data were included, these 7 canonical components together accounted for ∼91% of the data variability, with the first accounting for ∼31%. Even after removing the outlier, the canonical correlation remained strong (0.95 to 0.66), and we have illustrated relations with and without the outlier  had at least 1 significant canonical correlation, all of which were moderate to strong  using rarefied taxon read counts at the genus level from milk (source), infant oral (source), maternal oral (source), and maternal fecal . The milk microbiome was estimated to directly contribute ∼4.9% at day 2 and ∼0.3% at month 6 to the infant fecal microbiome (Streptococcus (Staphylococcus. This is similar to work performed by Costello and coworkers (Staphylococcus in oral samples collected from a small group of low-birthweight infants. We posit that the high relative abundance of Staphylococcus in the infant's mouth is likely associated with the high proportions of Staphylococcus in milk and on the mother's skin. With the frequent bi-directional interaction between the mammary gland and the infant's mouth during breastfeeding (This study relates and compares the membership and composition of microbiomes from maternal milk, feces, and oral samples, and infant feces and oral samples from the same mother-infant dyad at 9 time points in the first 6 mo postpartum. Samples from the different niches showed distinct clustering at the genus level as has been shown previously for other subsets of mother-infant pairs . Bacteritococcus , 47, 48 oworkers  in whichtfeeding , it is nOur data do not support our a priori hypothesis that the milk microbiome would most closely resemble that of infant feces. In fact, we observed that the milk microbiome was more similar to that of infant oral and, as time progressed, became more similar to both infant and maternal oral microbiomes. These results concur with previous studies  and prov16S rRNA database. Different sample types also had different average yields of sequence reads. In order to minimize the bias of having large variation in the sequencing depth between samples and sample types, we rarefied the read counts to a minimum threshold of 2,000 reads. Although we recognize that rarefying results in a loss of information, this approach has been suggested (16S rRNA gene. Therefore, we could not discern whether the DNA was from live or dead bacteria and were only able to characterize the majority of the sequence reads to the genus level. The results presented are also compositional in nature. Recently, Gloor and coworkers (There are several important limitations to this study. Although our sample size was larger than that of some others , 21, it uggested  to help oworkers  reviewedIn conclusion, microbial communities of the mother-infant epi-holobiont represent complex, collective microcosms that likely interact to maintain health in both mothers and infants , 71–73. nxy299_Supplemental_FilesClick here for additional data file.


Development of white spot lesions around orthodontic fixed orthodontic appliances is a common finding, especially in patients with poor oral hygiene. One of the conservative interventions for regression of these lesions is using chemical solutions. The current study aimed to compare the effectiveness of fluoride and amorphous calcium phosphate (ACP) on microhardness improvement of affected enamel.p< 0.05 was considered as significant.Forty-five intact human incisor teeth were selected and randomly divided into 3 groups of 15. Fluoride group, ACP group and artificial saliva group (control group). Inducing of white spot lesion was done by PH-cycling model. Samples of the first and second group were submerged into 0.05% fluoride and 0.05% ACP solutions respectively for one minute a day. The rest of the time, all specimens were put in artificial saliva, which was incubated in 37 °c temperature. Microhardness of specimens was assessed by Vickers microhardness test in three stages: 1: Baseline microhardness assessment that was done before induction of white spot lesion, 2: Secondary microhardness assessment that was done after induction, 3: Final microhardness assessment that was done after chemical treatment. The SPSS 11.5 software was used for statistical analysis and Microhardness of specimens in the fluoride and ACP groups had significantly improved after the treatment . In the control group, no significant improvements were observed. In final assessment, there were significant differences between the ACP and control groups, but no significant differences were found neither between the fluoride and ACP, nor the Fluoride and control groups.According to the current study, both 0.05% ACP and 0.05% fluoride solutions enhanced enamel micro-hardness in treatment of white spot lesion. Key words:Microhardness, amorphous calcium phosphate, fluoride, white spot lesion. White spot lesions or enamel decalcification is the clinical manifestation of early enamel caries and attributes to the prolonged retention and accumulation of dental bacterial plaque. The white appearance of these lesions is the result of different optical reflections due to mineral loss in the surface or subsurface enamel . A greatThe aim of the current study was to compare the effectiveness of 0.05% fluoride solution and 0.05% ACP solution on treatment of white spot lesion in an accurate lab trial.-Sample size calculation et al. , ACP solution (0.05% Ca3 (PO4)2 in 2% HPMC as a buffer) and modified Fusayama artificial saliva solution ( KCl (400 mg/l), NaCl (400 mg/l), NaH2PO4•H2O (690 mg/l), CaCl2•2H2O (795 mg/l), Na2S•9H2O (5 mg/l), KSCN (300 mg/l), and urea (1000 mg/l). Samples of the first and second groups were placed in 10 cc of fluoride rinse and ACP solution respectively for one minute a day on the vibrating machine (to resemble the mouth washing action). At other times, these specimens in addition to the third group specimens were kept in artificial saliva, which was incubated in 37˚C. After 10 weeks of performing this procedure for the groups, they underwent the final microhardness assessment.-Statistical analysis After evaluating the normality of data with the Kolmogorov–Smirnov test, multiple measurement tests were done to indicate any significant differences between baselines, secondary and final microhardness assessments in each group. In order to determine the differences of microhardness assessments between groups, the analysis of variance test (ANOVA) was used. Statistical Analysis was performed using SPSS 11.5 , and the P values less than 0.05 were considered as significant.p value = 0.049 and p value = 0.017 respectively). Further analysis showed that in fluoride group significant differences existed between secondary and final assessment , while in ACP group significant differences observed between baseline and final assessments and also secondary and final assessments . No significant difference was seen among baseline, secondary and final microhardness assessment of control group.et al. article revealed that use of CPP-ACP paste could treat white spot lesions in permanent teeth of patients, and supplementary application of 5% sodium fluoride varnish had no beneficial effects on regression of these lesions. They believed that fluoride therapy could result in hypermineralization of surface layer in presence of fluoride ion, which blocks entering of mineralizing ion to the subsurface area  and covering the ambient light. The bias of using QLF has been suggested to be removed by longer period of study with a greater sample size and identical baseline characteristics of the treatment and control group (White spot lesions are common problems in many orthodontic patients with fixed appliances, and different preventive measures have been suggested to reduce their incidence . Local face area . Kumar eotential . In a syotential . On the otential ,26,27. Aol group ,29. In tin-vivo investigations are needed. Effectiveness of fluoride in prevention of white spot lesions has been proved with strong evidence in well-designed systematic review (In the current study, intact teeth with no defects were selected and underwent the same protocol for white spot inducement. The microhardness assessment which is an accurate method for determining mechanical properties of carious lesion was used and compared between groups. Our investigation demonstrated that physicomechanical properties of teeth treated with both fluoride and ACP improved, and although this improvement was higher in the ACP group but no statistical difference was found. Despite these results, there is more important question to respond. Whether such different in microhardness improvement can lead to higher clinical outcome or not? The answer to this question is unclear and longitudinal c review . But thec review  as a proc review ,30. Orth Although there are investigations on the combined use of these solutions, we suggest a well-designed clinical trial to be performed to investigate the effectiveness of fluoride solution, ACP solution and combination of these two products.Fluoride solution and ACP solution enhanced the enamel microhardness of white spot lesions and it seems they are suitable products for treatment of white spot lesions.


Climate models predict a possible increase in the frequency of strong climate events such as El Niño-Southern Oscillation (ENSO), which in parts of the tropics are the cause of exceptional droughts, these threaten global food production. Agroforestry systems are often suggested as promising diversification options to increase farmers' resilience to extreme climatic events. In the Northeastern state of Bahia, where most Brazilian cocoa is grown in wildlife-friendly agroforests, ENSOs cause severe droughts which negatively affect forest and agriculture. Cocoa (Theobroma cacao) is described as being sensitive to drought but there are no field-studies of the effect of ENSO-related drought on adult cocoa trees in the America's; there is one study of an experimentally-imposed drought in Indonesia which resulted in 10 to 46% yield loss. In our study, in randomly chosen farms in Bahia, Brazil, we measured the effect of the 2015–16 severe ENSO, which caused an unprecedented drought in cocoa agroforests. We show that drought caused high cocoa tree mortality (15%) and severely decreased cocoa yield (89%); the drought also increased infection rate of the chronic fungal disease witches' broom (Moniliophthora perniciosa). Ours findings showed that Brazilian cocoa agroforests are at risk and that increasing frequency of strong droughts are likely to cause decreased cocoa yields in the coming decades. Furthermore, because cocoa, like many crops, is grown somewhat beyond its climatic limits, it and other crops could be the 'canaries in the coalmine' warning of forthcoming major drought effects on semi-natural and natural vegetation. Climate change is likely to affect global food production –3. AgricSpondias mombin and exotic Artocarpus heterophyllus), timber  or nitrogen fixation (such as native Plathymenia foliosa and exotic Erythrina spp). These agroforestry systems, called cabrucas in Bahia 1/t where N0 is the number of trees at the beginning of the interval, Nm the number of trees that died after one year (time t = 1 year). Cocoa trees were classified into 3 groups by MCCS technicians based on field characteristics: 1) ‘common’—Amazonian Amelonado including Marañon and Para the varieties most commonly found in Bahia; 2) ‘clones’, grafted or rooted cuttings of identified clones such as CCN51, TSH1188, PH16 or PS1319 selected by research institutes; and 3) hybrids, seed material produced in the 1960’s by CEPLAC mainly from hybridization of the ‘clones’ and other varieties.The number of dead cocoa trees was recorded in the middle of the second drought in April 2016 and compared to the number of live cocoa trees recorded in April 2015. Dead trees were recorded again in November 2017 and additional mortality was marginal (1.7%). The mortality rate of the cocoa trees . Potential yield was estimated using a conversion factor of 40 g of dry cocoa beans per healthy cocoa pod before the drought and 23 g of dry cocoa beans per healthy cocoa pod during after the drought (a relationship established for cocoa in the area). Both averages were calculated based on ripe cocoa pod sampling from Barro Preto farms respectively before the drought (May 2012) and at the end of the drought (May 2016). Approximately 30 pods were opened, fresh beans were fermented 5 days, sun-dried for about a week before being weighted.The number of cocoa pods on tree counted before the bi-annual harvests in the transects was used as a proxy to estimate an average potential yield per harvest. The number of cocoa pods  was counted on each tree before the harvest by the farmers in April 2015, April 2016, November 2016 and April 2017. Pod number on trees in the 800 mMoniliophthora perniciosa but also infrequently by Phytophthora palmivora) was assessed by recording the number of rotten pods on each tree. M. perniciosa primarily affects the leaves and vegetative development, but also impacts directly cocoa pods. The November 2015 harvest was not recorded for logistic reasons. Potential losses due to drought and disease were calculated by comparing the number of pods counted during harvests during and after the drought with the number of pods counted during harvest before the drought.The fungal infection rate . All statistical analyses were computed using R Stat version 3.4.1.When the study began in March 2015, NOAA reported an Oceanic el Niño Index (ONI) anomaly of > +0.5°C in Sea Surface Temperature (SST). ONI reached a maximum of +2.3°C in December 2015 and was > +0.5°C for 16 consecutive months until May 2016. The ENSO 2015–16 caused an abnormally strong drought in the cocoa region of Bahia in Brazil. Rainfall between August 2015 and August 2016 was 770mm (and 786 mm between August 2016 and May 2017); this is 53% lower than the 1621 (±71) mm yearly average for 14 years from August 2001. The months with the minimum rainfall were September and November 2015 with 6 and 7 mm per month respectively .Comparing the 30-day rainfall totals with the Potential Evapotranspiration (PET)  showed t2 = 0.63, p < 0.01). During the ENSO-related drought, calculated soil water content was at about zero for 4 months from mid-September 2015 to mid-January 2016 (Soil water holding capacity was about 86 (±13 SE) mm in the top 1000 mm of soil , it was ary 2016 .2 = 53.2, 2 d.f., P<<0.001). There was no relationship between shade tree density, either numbers or total basal area, and cocoa mortality  during the ENSO event as compared to <1% normally . In a further 5% (±1.2 SE) of the trees the large productive stems died but suckers remained alive. Cocoa mortality differed between groups 28% in hybrids, 22% in ‘common’ cocoa and 15% in ‘clones’  on the 31 farms, for the main harvest (of two per year) in April 2015 before the drought, was 242 (± 25) kg/ha. During the drought, in April 2016, the average potential yield was 26 (± 9) kg/ha—an 89% reduction . Nine moThere was no relationship between soil water holding capacity and yield loss. There was an effect of farm location (longitude) on yield loss .Witches’ broom was first recorded in Bahia in 1989 and has resulted in a big reduction of cocoa production since. At the beginning of the study (April 2015), the fungal infection caused about a 15% pod loss. However, during the drought the loss was much higher: 36% in April 2016 and 35% in April 2017 after the drought.Strong ENSO-related droughts decreased production in Bahia and Brazil as a whole , though -1) was the lowest recorded since 1990. At a country scale yield decreased from 400 kg ha-1 before the drought to 277 kg ha-1 after; in Para state yield decreased from 860 kg ha-1 to 490 kg ha-1, in Bahia state yield decreased from 300 kg ha-1 to 200 kg ha-1. Production for Barro Preto was not available for 2016–2017).Cocoa production in 2015–16 during the ENSO-related drought (277 kg haAlbizia ferruginea and cocoa-Antiaris toxicaria) in only one farm. The generality of this conclusion have been questioned by [To our knowledge, our study on the effect of drought on cocoa agroforests, is the first, in South America, based on natural drought and field assessments on farms. Despite cocoa being known as a drought-sensitive crop  publisheioned by ,45 who pGliricidia, to create a 13-month artificial drought [Gliricidia roots were found evenly distributed deeper into the soil (up to 250 cm depth). Water uptake for cocoa occurred in the first 30 upper cm of soil whereas water uptake for Gliricidia occurred from depths >30 cm [Gliricidia roots. The difference in the distribution of roots could explain the high mortality rate in cocoa trees and low mortality of other tree species in our experiment.In the only on-farm experiment on drought in Indonesia, plastic roofs were used over an eight-year cocoa plantation shaded by  drought . Most coStudies of the effect of previous ENSO events on cocoa plantations at regional scales in Asia, Africa and America were based on indirect data resulting from interviews and/or national statistical compilations of yield and planted area ,26,46,47Despite large uncertainty , severe -1). The difference could be due to one of several, non-mutually exclusive, reasons. Firstly, our farms may have been unusually affected by the drought as compared to the rest of the state of Bahia; we cannot test this but point out that our farms were a random sample from 333 farms in the Barro Preto region (160 km2) however this is only 0.5% of the cocoa region of the State of Bahia  and though the area sampled was large (16 km West to East and 10 km North to South) it is small compared to the ‘cocoa area’ of Bahia. Secondly, it could be that in wetter parts of the cocoa region of Bahia  the ENSO-related drought had little effect or even increased growth because in wetter areas, the potential reduction in yield due to some drought may be overwhelmed by the increased production resulting from increased solar radiation. Thirdly, drought may cause abnormally high leaf loss in shade trees, which has two effects it potentially somewhat reduces water use by the shade trees and it also lets more light through to the cocoa trees below, thus increasing yield .after the rooves were removed; there was no cocoa tree mortality [There seems to be only one other on-farm study of the effect of drought on cocoa yield—an experimental drought, simulating an ENSO event, in Indonesia, where rooves reduced rainfall by about 78% over 13 months, when the actual rainfall was 2937mm. In this experiment, there were no extended periods without rain and the ‘relative extractable water’ from the soil only reached close to zero for one month near the end of the experimental drought. Cocoa yield was only reduced by 10%, though interestingly yield was reduced by 45% ortality . By contThe direct effect of the drought (low water availability in soil) was to reduce the cocoa pod production and to kill many cocoa trees. Additionally, the drought could have had indirect effects on cocoa yield which were not measured during the experiment. For example, the drought could have affected the cocoa pollinator populations causing an indirect reduction in cocoa yield. In the artificial drought experiment in Indonesia, the results of hand-pollinations were not affected by the drought. However, pollination intensity was a main factor limiting yield in both roof and control plots . In BahiP. palmivora) in Bahia [In addition to the direct and indirect effects of drought reducing cocoa tree growth and pod production, drought was associated with an increase in disease infection rate . Rotten pods resulting from disease contributed significantly  to the reduction in the number of pods counted after the drought. This increase in infection rate was also observed in the artificial drought experiment in Indonesia [in Bahia  and in Nin Bahia . Fungal in Bahia , thus puhttp://www.arapyau.org.br blog/2016/01, online access: 25-07-2017). Exceptionally high infection rates of pests and diseases can also be responsible for high tree mortality, for example a change from heavily shaded agroforests to non-shaded systems resulted in insect attack in São Tome and Fernando Pó in the 1920’s [(Oncobasidium theobromae) in Malaysia in the 1990’s [Moniliophthora perniciosa does not kill trees). The high mortality of mature cocoa trees caused by drought is important because it will reduce yields for a minimum of 3–5 years until replacement trees become productive.Cocoa tree mortality, 15%, was exceptionally high for reasonably healthy old cocoa plantations, where normal annual tree mortality is usually <1% ,57. Highe 1920’s  and Vasce 1990’s . No lethModern clones selected for their drought-tolerance characteristic were not found in our plots. However, we showed that clones not necessarily selected for the drought were more resistant than hybrids or traditional common amelonado. These clones include e.g CCN51, CCN10, PS1319 and TSH1188, accessions often selected for their high yield and disease resistance in high input and low-shade conditions, which could partially explain their drought tolerance. Numerous drought-tolerant cocoa accessions have been identified ,60,61. HRecent ENSO-related drought has changed the balance of cocoa production between Brazilian states; Bahia used to produce 95% of Brazilian cocoa, but since the 1990’s, Bahian production has been declining mainly because of witches’ broom whereas production in Para state has been increasing considerably since it started in the 2000’s. In 2017 just after the drought, Para state became the most productive Brazilian state. The 2015–16 ENSO- drought reduced cocoa production in Bahia, which was already weakened by chronic fungal infection. Such changes in producing areas are common in cocoa and have been described as part of boom and bust cycles . In the World cocoa production is negatively affected by ENSO years and positively affected by la Nina years . HoweverThus, we have shown, for the first time on-farm, that a severe El Niño drought reduced cocoa production by 89% for the main (April) harvests in 2016 and 2017, and killed 15% of cocoa trees. Strong droughts are not uncommon in Bahia Brazil, but an eleven-months event with two successive droughts of 136 and 131 days (separated by 25 days) was unique. It is likely that such droughts will become longer and more frequent in the tropics and thus cocoa yields in such areas will be strongly reduced. Cocoa is an example of many crops grown somewhat beyond their normal climatic range; which are sensitive to drought and whose yields might be greatly reduced in future due to changed climates with stronger and more frequent droughts. Such crops could be the ‘canaries in the coal mine’ warning of problems to come due to increased intensity and frequency of droughts ,74, bothS1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S8 Table(DOCX)Click here for additional data file.S9 Table(DOCX)Click here for additional data file.


IntroductionWe conducted a survey to describe the practice characteristics of anesthesiologists who have passed the American Board of Anesthesiology (ABA) Pediatric Anesthesiology Certification Examination.Methodstheaba.org). Email contact information for these individuals was collected from departmental rosters, email distribution lists, hospital or anesthesia group profiles, manuscript author contact information, website source code, and other publicly available online sources. The survey was designed using Qualtrics , a web-based tool, to ascertain residency/fellowship training history and current practice characteristics that includes: years in practice, clinical work hours per week, primary hospital setting, practice type, supervision model, estimated percentage of cases by patient age group, and percentage of respondents who cared for any patient undergoing a fellowship-level index cases within the previous year. The invitation to complete the survey included a financial incentive - the chance to win one of twenty $50 Amazon gift cards.In July 2017, a list of anesthesiologists who had taken the ABA Pediatric Anesthesiology Certification Examination (hereafter referred to as "pediatric anesthesiologists") was obtained from the American Board of Anesthesiologists  and 962 complete survey responses were received . Over 80% (785) of respondents completed a pediatric anesthesiology fellowship. Of these, 485 respondents (50.4%) work in academic practice, 212 (22.0%) in private practice, 233 (24.2%) in private practice and have academic affiliations, and 32 (3.3%) as locum tenens or in other practice settings. The majority of respondents (64.3%) in academic practice work in freestanding children’s hospitals. Pediatric anesthesiologists in academic practice and private practice with academic affiliations reported caring for a greater number of younger children and doing a wider variety of index cases than respondents in private practice.ConclusionThe extent to which pediatric anesthesiologists care for pediatric patients - particularly young children and those undergoing complex cases - varies. The variability in practice characteristics is likely a result of differences in hospital type, anesthesia practice type, geographic location, and other factors.  The field of pediatric anesthesiology has evolved and grown substantially since it became an American College for Graduate Medical Education (ACGME) approved fellowship in 1997 [Until recently, no comprehensive list of pediatric anesthesiologists in the United States (US) existed. In 2015, our group created the first database of pediatric anesthesiologist demographic characteristics and geographic locations in the US [Understanding practice characteristics of pediatric anesthesiologists - the anesthesia practice types and hospital settings in which they work, categories of patients they care for, and for what types of procedures - are necessary for a better understanding of the spectrum of anesthesia care provided to pediatric patients in the US and is relevant for workforce planning, fellowship education, and outcome research. Therefore, we conducted a survey of the practice characteristics of anesthesiologists who passed the ABA Pediatric Anesthesiology Certification Examination. theaba.org" using the "Advanced Search" function. From October to December 2017, email contact information for these individuals was collected from departmental rosters, email distribution lists, hospital or anesthesia group profiles, manuscript author contact information, website source code, and other publicly available online sources. In July 2017, a list of anesthesiologists who registered for the ABA Pediatric Anesthesiology Certification Examination was obtained at "The Stanford University Administrative Panel on Human Subjects in Medical Research waived the requirement for written informed consent and approved the study.Survey designThe survey instrument was designed and distributed using Qualtrics , a web-based tool for creating and conducting online surveys. Survey questions were designed to determine residency/fellowship training history and current practice characteristics including years in practice, clinical work hours per week, primary hospital setting, practice type, supervision model, estimated percentage of cases by patient age group, and percentage of respondents who cared for any patient undergoing a fellowship-level index cases within the previous year. Fellowship-level index cases are an indicator of complex pediatric anesthesia care and include airway surgery (excluding tonsillectomy and adenoidectomy), cardiac surgery with cardiopulmonary bypass, cardiac surgery without cardiopulmonary bypass, craniofacial surgery, intra-abdominal/intracavitary surgery, intracranial neurosurgery, intrathoracic non-cardiac surgery, major orthopaedic surgery, neonatal emergency surgery, and solid organ transplant .Anesthesiologist sex was imported from an existing database. The years in practice was estimated based on the time of primary board certification in anesthesia until the survey date. Survey responses were retained for those who fully completed the demographics and practice characteristics sections of the survey. Respondents and non-respondents who registered for the ABA Pediatric Anesthesiology Certification Examination but did not subsequently pass were excluded from the analysis as to include only anesthesiologists with ABA Pediatric Anesthesiology Certification.The survey was first distributed to 20 members of the authors’ department and revised based on their suggestions for clarity and for improvement. In January 2018, the revised survey was emailed to all pediatric anesthesiologists whose email address had been identified (Supplement 1). The invitation to complete the survey included a financial incentive - the chance to win one of twenty $50 Amazon gift cards.Two reminders were sent to survey non-respondents within the next 30 days. The survey was closed six weeks after the initial email.There were 3,492 anesthesiologists who had taken or were registered for the ABA Pediatric Anesthesiology Certification Examination in July, 2017. Surveys were sent to 2,681 individuals whose emails were identified; 962 (35.9% of those emailed) complete survey responses were received  work in an academic practice, 212 (22.0%) in private practice, 233 (24.2%) in private practice and have academic affiliations, and 32 (3.3%) as locum tenens or in some other practice setting.Pediatric anesthesiologists in academic practice reported working fewer clinical hours than those in private practice or in a private practice with an academic affiliation. The majority of pediatric anesthesiologists (64%) in academic practice work in a freestanding children’s hospital at least 50% of the time, whereas only 17% of those in private practice, and 47% of those in private practice with an academic affiliation do the same. The majority of anesthesiologists in academic practice supervise residents (96.7%), fellows (80.8%), certified registered nurse anesthetists , and non-anesthesia trainees (58.6%) whereas the majority in private practice supervise CRNAs only Table . More thPediatric anesthesiologists in academic practice indicated that the majority of their patients were five years old or younger 52.7%) versus 33.2% for those in private practice, and 45.3% for those in private practice with an academic affiliation. All practice characteristics, stratified by anesthesia practice type, are shown in Table .7% versuThe results of this survey illustrate the practice characteristics of anesthesiologists who successfully completed the ABA Pediatric Anesthesia Certification. An understanding of their practice characteristics is important for workforce planning, fellowship education, outcome research, and optimizing the care of children.The results of this survey suggest that, in general, respondents in academic practice or private practice with academic affiliations care for a higher percentage of younger patients as well for as patients undergoing a wider variety of fellow-level index cases, than those in private practice. Over 90% of all respondents, regardless of practice type, reported caring for patients undergoing at least one index-level case type in the past year. On the other hand, nearly one in 10 respondents in private practice (or one in 20 respondents in academic practice or academic practice with an academic affiliation) reported caring for no patients undergoing an index case within the preceding year. This issue is particularly timely, given the potential effect of the American College of Surgeon's Children's Surgery Verification Quality Improvement Program on the distribution of pediatric surgery in the US. It is possible that with regionalization of pediatric surgical procedures, the number of pediatric cases per pediatric anesthesiologist at non-tertiary care children's hospitals will decrease. This raises questions about how anesthesiologists will maintain clinical competency given a potential reduction in the number of children undergoing care in the community hospital setting.There are certain limitations that should be considered when interpreting the results of this survey. The survey describes the practice characteristics of anesthesiologists who passed the ABA Pediatric Anesthesiology Certification Examination; the respondents may be biased toward the academic end of the practice spectrum, where certification is often required. In addition, there are many anesthesiologists who did not take the ABA Pediatric Anesthesiology Certification Examination who nevertheless care for children routinely. Anesthesiologists who trained before the advent of time-limited anesthesiology subspecialty boards hold board certifications that include all subspecialties. The survey misses a large number of legitimately qualified practitioners who hold "board certified" status in the field of anesthesiology - inclusive of pediatric anesthesiology. In addition, the survey response rate was low (35.9%), thus raising the possibility of bias in the results. Although respondents and non-respondents appeared similar in terms of sex, years in practice, and geographic location, they may differ in unobservable ways, thus potentially limiting the generalizability of the results. Nevertheless, this survey gives us an improved understanding of the practice characteristics of those pediatric anesthesiologists with ABA Pediatric Anesthesiology Certification.The practice characteristics of pediatric anesthesiologists are now better understood. The extent to which pediatric anesthesiologists care for pediatric patients - particularly young children and those undergoing complex cases - varies. The variability in practice characteristics is likely a result of differences in hospital type, anesthesia practice type, geographic location, and other factors.


Tachypleus tridentatus and Carcinoscorpius rotundicauda. Three types of hemocytes were distinguished respectively: the granular cell, the semi-granular cell and the hyaline cell by transmission electron microscopy, while three hemocyte subpopulations  were classified by flow cytometry. Hyaline cell was the major cell type with the highest nuclear-cytoplasmic ratio and granular cell and semi-granular cell showed lower ratios. Immune parameters of hemocytes in horseshoe crabs were investigated by flow cytometry. Different hemocyte subpopulations respond for diverse functions. Lysosomal contents and hemocyte mortality in Gate 3 cell subpopulation were higher than that in other subpopulations, while reactive oxygen species, phagocytosis and non-specific esterase, in Gate 1 cell subpopulation, were higher than those in other subpopulations. The hemocyte types between the two species had no significant differences in staining or morphology.In present study, transmission electron microscopy and flow cytometry were utilized to investigate the classification, characterization and immune functions of hemocytes from horseshoe crab,  T. tridentatus is vulgarly named as the Asian3, Chinese4 or Japanese horseshoe crab5 and C. rotundicauda is also called Asian3 or mangrove horseshoe crab6. The two species are distributed from East Asia and Southeast Asia12. In China, these two species live in the South China Sea and the East China Sea13, especially in some areas like Hong-Kong14 and the Beibu Gulf15.The horseshoe crab is a living fossil animal, belonging to Arthropoda, Chelicerata, Merostomata, Xiphosura. There are merely four species surviving in the worldT. tridentatus has been regarded as a protected species owing to being endangered since 1928, but its population remains to be under threat16. The similar status also exists in China, for example, in Hong Kong the horseshoe crab population is also decreasing15. As an invertebrate animal, horseshoe crab only possesses of innate immune system19 and does not have adaptive immune system20. Hemocyte plays an important role in innate immunity as shown in many invertebrate animals23. Many studies on the blood of horseshoe crabs indicated that some elements in the blood are capable of eliminating some virus or foreign particles26, equivalently emphasizing the importance of hemocytes to immunity in horseshoe crab. Therefore, basic information on hemocytes in Chinese horseshoe crab T. tridentatus and mangrove horseshoe crab C. rotundicauda can provide insights for their health and immunology.In Japan, Cancer borealis and Cancer pagurus27, the pearl oyster Pinctada fucata28, the Atlantic jackknife clam Ensis directus29, the Pacific oyster Crassostrea gigas23 and the zebra mussel Dreissena polymorpha22 using electron microscopy or flow cytometry. Using flow cytometric technique, the hemocyte immune parameters, such as total hemocyte counting (THC), phagocytosis (Pha), non-specific esterase (Est), lysosomal content (Lyso), hemocyte mortality (Hm) and reactive oxygen species (ROS) can be evaluated as well. Hemocyte parameters have been investigated in the pearl oyster Pinctada fucata28, the pacific oyster Crassostrea gigas23 and the zebra mussel Dreissena polymorpha22 by flow cytometry. The hemocyte types of cultivated Chinese horseshoe crab were classified into granule flattened cells, degranulated flattened cells and contract flattened cells by just light microscopy30. Also, the information on hemocytes of horseshoe crabs (T. tridentatus and C. rotundicauda) were investigated using optical microscopy and scanning electron microscopy, and the preliminary hemocyte classification has been proposed31. However, the inside features and immune parameters  of hemocytes in the two Asian horseshoe crab species have not been reported well.Three types of hemocytes have been identified in the two edible crabs Portunus trituberculatus showed differences32. A comparative study of the hemocyte properties showed different functions between Mytilus edulis and Aulacomya ater33. A comparison between the mussel Mytilus galloprovincialis and the oyster Crassostrea gigas was made to ascertain which hemocyte possesses a higher resistance to the macrooganism infection34.Some comparisons of hemocytes among/within species have been reported previously. The immune functions of hemocytes in different breeding generations of the swimming crab T. tridentatus and mangrove horseshoe crab C. rotundicauda on the immune parameters by flow cytometry. These new findings on horseshoe crab hemocytes can provide useful references for studying immunology of these two horseshoe crabs species.In the present study, transmission electron microscopy (TEM) and flow cytometry were used to view the interior morphology and classify the sub-populations of the hemocytes. In addition, THC, Pha, Est, Lyso, ROS and Hm were measured in two horseshoe crabs by flow cytometry. The study is the first comparison between Chinese horseshoe crab The ultra-structures of the hemocyte were observed using TEM, dividing the cells into three types  Fig. . GCs werT. tridentatus was more centralized than C. rotundicauda  of MTT, FCR and MCR (50000–55000 mL−1) were significantly (p < 0.05) lower than FTT , male T. tridentatus (MTT) and female C. rotundicauda (FCR), male C. rotundicauda (MCR), were classified, according to the criteria of cell size (FSC) and the cell complexity  and divided into granular cells, semi-granular cells and hyaline cells. According to the number and size of granule, the invertebrate hemocytes could be easily to classify35. Three main types of hemocytes were characterized: granular cells, semi-granular cells and hyaline cells in two horseshoe crabs, whose hemocyte types are similar to some other invertebrates, including some crustaceans37. Besides, the hyaline cells possessed a big nucleus and a few large granules among the Chinese horseshoe crab and mangrove horseshoe crab as some other shellfishes38. According to the method of optical microscopy, granular cells, semi-granular cells and hyaline cells with different shapes as exhibited were typed for the hemocytes of Chinese horseshoe crab and mangrove horseshoe crab31, which are similar with our results. For the two horseshoe crabs, the types and features of hemocytes were similar, and the similar phenomena were also showed in other studies39. Besides, hyaline cells were unique contrasting to other cells, which is tally with the study in cultivate horseshoe crab before30. Previously, scanning electron microscope) also has been used to view the morphology of the hemocytes in horseshoe crab31. Based on the different size and varied numbers of wrinkle on the surface, the hemocytes were classified into three types: granular cells semi-granular cells and hyaline cells31.Until now, hemocyte classification in aquatic invertebrate has not reached a uniform standard, but most agree on the classification of granular cells and hyaline cells40, total hemocyte counts (THC) were determined in the male and female of the two horseshoe crab species. Among the two species with female and male, the similar results were obtained from the male Chinese horseshoe crab and both male and female of the mangrove horseshoe crab, while the female Chinese horseshoe crab showed some differences. Perhaps different species within the genus as well as different genders can show some differences in such parameter. For example, THCs between the white shrimps Litopenaeus vannamei and Litopenaeus stylirostris were quite different42. THCs were also analyzed in two edible crabs, the Jonah crab Cancer borealis and the brown crab Cancer pagurus, and there were some differences between the two crab species27.As an indicator representing the immune functions of hemocyte44, three23 and four45 types. FCS and SSC are sometimes not reflecting actual FCS/SCC in crustaceans by FCM46, thus the matches between microscopic images and FCM may not be reached exactly. In the present study, hemocytes of the Chinese horseshoe crab and mangrove horseshoe crab, for males and females, were classified into three groups . However, the proportion of each type of cell shows differences between two horseshoe crabs.In present study, hemocytes also were analyzed by flow cytometry and several indexes were obtained. Usually, the number of hemocyte types varies from species to species. Base on this method, hemocytes in some shellfishes were classified into two47. The phagocytosis of hemocytesis poorly defined, it is possible that different types of hemocytes in invertebrates possess different capacities in phagocytosis48 or some hemocytes were not phagocytes49. In the present study, it is obvious that the G1 cells  with less granular were the main phagocytes, whereas G2 and G3 cells had lower phagocytosis, and in the study of Mats50, the dominance of hyaline cells in phagocytosis was also observed. While the Chinese mitten crab Eriocheir sinensis43 showed a different result that G3 cells (possible granulacytes) were the main phagocytes. In our study, G1 cells of the four kinds of horseshoe crab were the main immunocompetent hemocytes, if phagocytosis activity was considered as the major indicator of immunity50. Lacking of intracellular prophenoloxidase, hyaline cells could be stimulated by the extracellular prophenoloxidase system, and degranulation has deeply connection with recognition function to foreign objects51. The similar result of the phagocytosis parameter was also showed in the mussel Mytilus edulis33.Phagocytes play a vital role in the innate immune responses in invertebrates52, mussels54, and clams55 has been extensively reported using flow cytometry. As a kind of hydrolase enzyme, it hydrolyzes some choline esters. This enzyme thus plays an irreplaceable role in the immune defense of marine invertebrates57, and the mortality of hemocytes increases when esterase activity decreases according to some studies58. In some studies, the granular cells showed higher esterase activity than hyaline cells and were regarded to the major cell in defense52, indicating the importance of its granular cells with esterase activity59. This was the first time for studying esterase of hemocytes in the Chinese horseshoe crab and mangrove horseshoe crab. For both species, the esterase in G3 cells was higher than that in G1 and G2 cells. That is to suggest that cells with more granules should be more active in esterase activity. Semi-granular cells possess stronger ability for exocytosis, playing an important part in immunity60.The esterase activity of marine invertebrates, like shrimps61, is regarded as an important mechanism for microbicidal capacities62. ROS is usually used to determine immunity in marine invertebrates, such as bivalves63 and crustaceans64. When hemocytes were stimulated (like exposure in vitro), respiratory burst occurred and toxic reactive oxygen species were released66. The most significant substances include superoxide anion (O2−), hydrogen peroxide (H2O2), singlet oxygen (1O2) and hydroxyl (OH)67. In the present study, the ROS production of G3 cells was higher than other hemocytes. The ROS of granulocytes was also higher than that in hyaline cells in the crab Eriocheir sinensis43. In the hemocytes of Crassostrea gigas68, higher ROS production was detected in granular cells.ROS production, induced by phagocytosis in the process of elimination69. Therefore, lysosome plays key roles in host defenses of microorganisms70. In the inactivation of invading microbes, lysozyme participates among the hemolymph constituents71. Many studies on hemocytes of marine invertebrates measured lysozyme in hemocyte, such as crabs43, oysters72 and mussels49. It was found in the green-lipped mussel P. viridis40 that the granular cells contained higher lysosomal content than other groups of hemocytes. Similarly in the present study, lysosomal content in G3 cells was higher than that in G1 cells in the two horseshoe crab species. Cytosolic lysosomes release various enzymes in vesicles, which combines with the production of ROS to destroy the foreign particles74. Meanwhile, there were some differences in G1 cells among the four kinds of horseshoe crabs, although all of them had lower lysosomal content.Lysosome is an organelle in cells, and lysozymes released from lysosome can non-specifically kill microorganisms by hydrolyzing cell walls75, thus hemocyte mortality is a useful indicator for immune system after being stressed in marine invertebrates76. There were some studies of hemocytes mortality in mussels63 and clams55. From the present study, hemocyte mortality was lower in G3 cells and higher in G1 cells. Combined with the lysosomal content, higher lysozyme safeguarded lower mortality in G3 cells, and in G1 cells lower lysozyme caused higher mortality, likely that lysosome is relevant to hemocyte mortality tightly owing to the high phagocytosis of G1 cells  hydrolyzing foreign objects after endocytosis72. For example, in the green-lipped mussel P. viridis40, the mortality of hyaline cells was higher than granular cells, indicating that hyaline cells were not positive as granular cells in immune defense. It is possible that different subpopulations are responsible for different functions48.The cellular mortality is usually accompanied by necrotic and apoptotic courses77. Crustacean hemocytes play important roles in the host immune response including recognition, phagocytosis, melanization, cytotoxicity and cell-cell communication78. Classification of the hemocyte types in decapods crustaceans is based mainly on the presence of cytoplasmic granules in hyaline cells, semi-granular cells, and granular cells79. Studies on crustacean have shown that different types of hemocytes play different roles in immunity80. Each cell type is active in defense reactions, for example, in crayfish, the hyaline cells are chiefly involved in phagocytosis, the semi-granular cells are the cells active in encapsulation, while the granular cells participate in storage and release of the prophenoloxidase and cytotoxicity78.Same as other crustaceans, the immune system of horseshoe crab lacks of immunoglobulin and removes exogenous object and pathogen through phagocytosis activity, esterase activity, ROS production and lysosome, belonging to innate immuneC. rotundicaud and T. tridentatus possess many morphological and functional characteristics as other marine invertebrates, some immune parameters in every types of hemocyte were different to other invertebrates. More studies on immune functions of different subpopulations of hemocytes in horseshoe crab should be further investigated.Apart from those, hemocytes in horseshoe crab need more deeply study. This study has demonstrated that the hemocytes of T. tridentatus  and C. rotundicauda , were collected from the Beibu Gulf, a semi-enclosed sea (17°00′ to 21°45′N and 105°40′ to 110°10′E). During the two-week period of acclimation, 12 T. tridentatus  and 12 C. rotundicauda  were fed on the same amount of Ruditapes philippinarum, once daily (17:00 h) before experiment, and no horseshoe crabs died. During the acclimation, water temperature was 26 ± 1 °C, pH was 8.1, salinity was 32 ± 1‰, dissolved oxygen (DO) was maintained at 6–8 mg L−1 and photoperiod was 12D:12 L with the light period from 06:00 to 18:00.Wild adult horseshoe crabs, −1) heparin81.After the adaptation period, six horseshoe crabs of each gender were sampled to obtain hemolymph for both species. Hemolymph was collected from the joint between bent breastplate and plastron using a 1.0 mL plastic syringe with 22 G needle and all samples obtained were stored in tubes with pre-chilled  in 0.1 M cacodylate buffer (pH 7.2) at 4 °C for one hour. Samples were douched in the same cacodylate buffer and distilled water, dehydration in ethanol solutions of six different grades  and transferred into acetone. Later before embedding, the samples were infiltrated gradually in resin. Ultrathin sections (60–90 nm) were made by using a Leica Ultracut UCT ultra microtome (Austria). Ultrathin sections were mounted onto coated collodion with 100-mesh copper grids. A section was dyed with 2% uranyl acetate aqueous solution for fifteen minutes before stain with Reynold’s lead citrate for ten minutes. Sections were observed using a FEI/Philips Tecnai 12 BioTWIN transmission electron microscope (Netherlands) operated at 80 kV.Five mL of hemolymph were prepared in the solution of 4% paraformaldehyde, 0.3 M sucrose in a 0.1 M cacodylate buffer (pH 7.2) and 2.5% glutaraldehyde for 10 minutes. Then, prepared cells were centrifuged at 400 g at ambient temperature for 10 minutes and the supernatant was discarded. The pellets were resuspended and encased in 3% molten agar. Then the embedded cells were whittled into 1 mmAn electronic particle counter/size analyser  was used to evaluate the hemocyte concentration in hemolymph. The size frequency distribution and hemocyte concentration (number of cells per milliliter) were determined. Before sample running, 0.5 mL of hemolymph was added into 9.5 mL of Isoton® II solution to crank out the mixture, and every time 1000 µL of the mixed solution were counted.® 10.0 software.Recently collected hemocytes within unwrought crude hemolymph were analyzed for immune parameters by a BD FACS Calibur flow cytometer equipped with an air-cooled argon laser and offers a laser excitation at a spot of 488 nm. A FSC limitation (>150) was defined to eliminate bacteria and cell debris. Datum was described as cell cytograms pointing the granularity , the proportional size  and the fluorescence channels identifying with the markers used. For each hemolymph sample, 20000 events were obtained in total, while the flow rate was corrected to maintain the whole events below 300 every second. The fluorescent frequency distribution column diagram of the hemocyte population was subsequently acquired. The type of fluorescence channel relied on the parameter monitored: hemocyte mortality was evaluated by FL2 (greenish orange emission for 585 nm), while reactive oxygen species (ROS), enzymes and phagocytosis were measured using FL1 (green emission for 530 nm). Data were analyzed by FlowJo−1 was added into 400 μL hemolymph, and the mixture was incubated for 30 minutes in the dark at 4 °C before analyzed by flow cytometry. Hemocyte mortality was analyzed as the percentage of hemocytes showing PI fluorescence relative to the whole hemocyte counts.Hemocyte mortality was detected by propidium iodide (PI), which is a type of fluorescent dye that only enters and stains dead hemocytes. 10 μL of solution of PI (Siama Aldrich) at a concentration of l mg·mL82. 400 μL of hemolymph was incubated for 1 hour at ambient temperature in the dark with 10 μL of the Fluorospheres® carboxylate-modified microspheres with 1/10 dilution . Finally, the fluorescent beads concentration was 108 mL−1, and the beads/hemocytes ratio was 100/1.Phagocytosis was detected using fluorescent microspheres as a phagocytic goal by an in italic assay, and estimated as the percentage of cells having internalized at least three fluorescent beadsActivity of non-specific esterase was detected using non-specific lip soluble ground substance fluorescein diacetate . FDA stock solutions (0.04 mM) were prepared in dimethyl sulphoxide (DMSO) and reserved at −20 °C. Working solutions of FDA (400 μM) were drew up by 1/10 diluting the stock with strained and sterile seawater. 400 microliters of hemolymph with 2 μL FDA solution was incubated in the dark for 15 minutes at ambient temperature. The percentage of cells expressing enzymatic activity was defined based on the percentage of fluorescent cells among all cells.The cellular reactive oxygen species (ROS) were measured by 2′7′-dichlorofluorescein diacetate . DCFH-DA, a nonfluorescent fluorescein analogue, can disseminate into hemocytes, and then is hydrolysed into 2′,7′-dichlorofluorescein (DCFH). Enzymatic activity was defined based on fluorescent cells among the whole cells. A 10 mM DCFH-DA stock was added to DMSO and is generally diluted to 10% in strained sterile seawater as a working fluid. Each analysis required mixture with four hundred microliters of hemolymph and 4 μL of DCFH-DA, and the mixture was then incubated in the dark for 15 minutes at ambient temperature.® Yellow HCK-123 . 400 μL hemolymph mixed with 1 μL of LysoTracker were incubated took place at ambient temperature for two hours in the dark and the reaction was ceased on ice. LysoTracker fluorescence in the hemocytes was described in arbitrary units (A.U.).Lysosomal content was measured by a merchant LysoTrackerPrior to the analysis, data were checked for homogeneity of variance with Levene’s test and normality with the Shapiroe-Wilk’s test using statistical software SPSS 18.0. Percentage data were arcsine transformed. One-way analysis of variance (ANOVA) and Tukey’s test were used to compare the differences of immune parameters among granular cell, semi-granular cell and hyaline cell identified by flow cytometry. For all analysis, the results are expressed as the means ± SD of the data and significant differences were known as p < 0.05.


Candida albicans causes two types of major infections in humans: superficial infections, such as skin and mucosal infection, and life-threatening systemic infections, like airway and catheter-related blood stream infections. It is a polymorphic fungus with two distinct forms  and the morphological plasticity is strongly associated with many disease causing proteins. In this study, 137 hyphae associated proteins from Candida albicans  were collected from different sources to create a Protein-Protein Interaction (PPI) network. Out of these, we identified 18 hub proteins  that were the most important ones in hyphae development. Ontology and functional enrichment analysis of these proteins could categorize these hyphae associated proteins into groups like signal transduction, kinase activity, biofilm formation, filamentous growth, MAPK signaling etc. Functional annotation analysis of these proteins showed that the protein kinase activity to be essential for hyphae formation in Candida. Additionally, most of the proteins from the network were predicted to be localized on cell surface or periphery, suggesting them as the main protagonists in inducing infections within the host. The complex hyphae formation phenomenon of C. albicans is an attractive target for exploitation to develop new antifungals and anti-virulence strategies to combat C. albicans infections. We further tried to characterize few of the most crucial proteins, especially the kinases by their sequence and structural prospects. Therefore, through this article an attempt to understand the hyphae forming protein network analysis has been made to unravel and elucidate the complex pathogenesis processes with the principal aim of systems biological research involving novel Bioinformatics strategies to combat fungal infections. The genome sequences of different Candida species have indicated that many of the genes involved in yeast to filamentous transition are evolutionarily conserved C. albicans are homolog to other Candida species C. albicans shows greater expansion in the number of genes relative to most of the other Candida species belonging to the same family. Consistent with this thought, less pathogenic Candida species have reduced the ability to produce virulent factors that are required for adhesion and invasion in the host cell in comparison to pathogenic hyphae forming C. albicansC. albicans, as it helps by promoting tissue penetration in host epithelial and endothelial cells and also avoids host immune system [C. albicans shows morphological plasticity and the transition from yeast to filamentous form in the host is a critical virulence determinant of infections C. albicans and the role of virulent proteins to elucidate the complex pathogenesis processes.itaniae) , 21 and e system , 26. C. C. albicans phenotypic plasticity by conversion from yeast to hyphae formation and development. We used Saccharomyces cerevisiae, a fungal model organism as a control for the validation of interactions and different aspects related to Candida hyphae forming proteins. With the limitation of antifungal drug availability and enhancing populations of susceptible patients, it is essential to understand the mechanisms of hyphae formation in order to develop new strategies for treating candidiasis. We have tried to infer the essential proteins and their regulation through the network analysis, which will be highly beneficial to understand the resistance mechanisms as well as for further development of anti-fungal therapy.In this work, we have implemented several bioinformatics and systems biology approach combining text mining methods to analyze and interpret the Protein-Protein Interaction (PPI) network of proteins that are involved in 22.1C. albicans were collected from literature and Candida Genome Database (CGD). The CGD is a well-organized repository containing various kinds of genomic, proteomic, morphology and annotation related information from four Candida species including C. albicansC. albicans revealed that more than 70% of enlisted proteins are uncharacterized. Orthology analyses of these proteins were carried out by using NCBI BLAST Candida species and Saccharomyces cerevisiae. We used Saccharomyces cerevisiae, a fungal model organism, as control for the validation and analyses of different aspects of protein-protein interactions (PPI) amongst the hyphae forming Candida proteins. All the PPI data were retrieved from several online resources  and literatures [Proteins that are known to participate in hyphae formations in eratures , 34, 35.2.2The total PPI datasets were arranged in a network form by Cytoscape 3.6.1 2.3k, clustering coefficient C(n), Betweenness centrality BC(n) and Closeness centrality CC(n). Degree k defines the number of directly connected neighbors of a node. The clustering coefficient C(n) is a measure of the degree of a node that has a greater probability to cluster together in the network. Similarly, Betweenness centrality BC(n)is the relative frequency of all paired shortest paths of a particular node in a network gives the information about the extent of interactions that a node mediates in a network. Closeness centrality CC(n) shows the spreading of information of a node and is defined as the reciprocal of the average shortest path length in a network [CC(n) would represent the strength of the interactions. A shorter path length would mean a stronger interaction. Another important attribute is the node degree distribution P(k), which aids to declare whether a network is random or scale-free, and is calculated by fitting the power law using equation P(k) has been used to evaluate the robustness of the network Topological analyses were performed by employing “Network Analyzer” plugin of Cytoscape  network , 42, 43.2.4k. Additionally, the Cytohubba plugin of Cytoscape was used to identify the hub proteins/nodes from the network Hub proteins are the ones which have the maximum number of interacting partners in a network 2.5Molecular complex detection (MCODE) plugin of Cytoscape was used to identify highly connected local sub-networks from the total PPI network 2.6C. albicans were retrieved and subjected to ClueGO analyses. Finally, two-sided hypergeometric test (enrichment/depletion), with Bonferroni steps down for pV correction at 0.05 significance level  and kappa score of 0.4 were set as threshold to analyze the network.Functional annotations of top scoring clusters were performed online at DAVID Bioinformatics Resources server 2.7http://services.mbi.ucla.edu/SAVES.Amino acid sequence of Candida Chk1 kinase domain  was downloaded from Uniprot (ID: Q5AHA0), and subjected to the template-based threading and modeling server I-TASSER 33.1C. albicans. For this, we collected the information of a total of 137 potent proteins having association with hyphae formation and regulation from published literatures [PPI network analysis is a crucial approach towards the understanding of the mechanisms of complex biological reactions and their possible outcomes. In the present work, we focused on building comprehensive network of proteins to analyze their modes of interactions leading to hyphae formation in eratures , 52, 53 3.2b’ was found to be -0.833, which signifies its reliability and the importance of hubs in the network , network centralization, and network density were found to be 0.325, 0.280, and 0.077, respectively. The maximum value of average clustering coefficient (0.325) was observed to justify the network with impressive measurement of nodes to be clustered together. Similarly, the network centralization score signified the importance of each node with good resemblance to the ratio of actual connections to the total possible connections within a network (density). The number of shortest paths was 18632 in the Candida hyphae PPI network, which would indicate that the nature of connectivity of the proteins was relatively high. The result also reveal that the transmission of biological information in the network was achieved through only a few steps as these proteins were involved in hyphae formation in the species by responding to various physiological and environmental clues. Similarly, the value of the degree centrality could identify the important nodes in the network on the basis of number of interactions, which were distinguished in terms of shared pathways or biological processes. The distribution of closeness centrality CC(n), and Betweenness centrality BC(n) were presented in CC(n) of a node in a network gives the idea about information that is passed from one node to another by measuring the number of shortest paths passing through the nodes from a PPI network BC(n) analysis of nodes revealed the proteins that could act as bridges or connect distant proteins together in the network.Other various parameters of the PPI networks, such as clustering coefficient 3.3BC(n) presuming these two to act as bridges, or bottlenecks, and were necessarily responsible for keeping the other nodes of the network intact , Serine/threonine-protein kinase , Cellular response to starvation , Filamentous growth  and cAMP-mediated signaling .The sub-networks generated by MCODE plugin were ranked on the basis of their confidence score, which is an indicator of their likeliness to form real protein complexes P value: 1.3E-16), small GTPase mediated signal transduction , filamentous growth , small GTPase superfamily , nucleotide-binding  and fungal-type cell wall organization . Similarly, the cluster 3 was detected as the largest one and associated with MAPK signaling pathway , two-component regulatory system , kinase , signal transduction histidine kinase , phosphoprotein , and cellular response to farnesol . Total 12 previously identified hub proteins were rediscovered in these clusters, where Cyr1, Ras1, Tpk2, Tpk1, Cdc28, Hsp90 were detected in cluster 1, Hog1 & Cdc42 in cluster 2, and Pbs2, Cdc24, Rim101 & Bem1 in cluster 3.On the other hand, the proteins in cluster 2 were annotated with versatile functions such as MAPK signaling pathway - yeast , regulation of filamentous growth of a population of unicellular organisms (GO:1900428), cellular response to oxygen-containing compound (GO:1901701), single-species biofilm formation (GO:0044010), interaction with host (GO:0051701), signal transduction by protein phosphorylation (GO:0023014), negative regulation of filamentous growth of a population of unicellular organisms (GO:1900429), cellular response to abiotic stimulus (GO:0071214), filamentous growth of a population of unicellular organisms in response to chemical stimulus (GO:0036171), positive regulation of response to external stimulus (GO:0032103) , purine nucleoside binding (GO:0001883), protein serine/threonine/tyrosine kinase activity (GO:0004712), protein kinase regulator activity (GO:0019887), calcium ion transmembrane transporter activity (GO:0015085), MAP kinase activity (GO:0004707), dolichyl-phosphate-mannose-protein mannosyltransferase activity (GO:0004169) and actin binding (GO:0003779) , hyphal tip (GO:0001411), fungal-type cell wall (GO:0009277) , adhesion involved in single-species biofilm formation (GO:0043709) cell adhesion involved in single-species biofilm formation (GO:0043709), negative regulation of response to stimulus (GO:0048585), and regulation of filamentous growth of a population of unicellular organisms in response to pH (GO:1900741)  . From ou3.7Candida, and a non-human homolog. structure of Candida Chk1 kinase domain is shown in The top 20% of nodes having higher degrees, functional enrichment analysis and multiple morphological features reveals that the proteins with kinase activity were predominant. Among them Hog1, Ssk2, Pbs2, Chk1, Cdc28, Tpk2, Pkc1& Cla4 were the leading kinases showing variable roles in candida hyphae formation. Sequence alignment of this large family of kinase proteins showed <30% of sequence similarity among themselves a. Additi4C. albicans through its hyphae formation. The proteins responsible for hyphae formation are considered as the integral components for the major virulence strategy of C. albicans. Expressions and the interactions of these proteins are believed to exert various cellular functions, adaptation to adverse conditions, and inducing pathogenesis. Network analysis in the article is an informative tool to direct novel experimentation to provide further insight into the mechanism of pathogenesis and virulence of C. albicans. Hence, the understanding of these PPIs is essential to study the pathogenic mechanisms in C. albicans and also for developing new therapeutic strategies. In this work, we constructed a network of 137 proteins that have role in hyphae development and studied their functions by network topology, hub, clustering, and functional enrichment analysis.Recent studies have revealed the pathological importance of Topological analysis confirmed our network as biologically scale-free and robust. From the average clustering coefficient and number of shortest path values, it was ascertained that the connectivity among proteins were very high. Centrality analysis of our predicted hyphae network yielded information regarding hubs which further helped to identify the hub proteins using CytoHubba. Out of the total 137 proteins, 18 possesses more than 20° among which Act1, Cdc28, Cdc42, Cla4 & Cyr1 were identified as interactome with the largest connections and were also involved in the promotion of hyphal growth, whereas Hog1, Hsp90, Pbs2, Pkc1 &Tpk1 were recognized as major suppressor of hyphal growth with the largest number of connections.C. albicans.Modular analysis by MCODE produce three large clusters of proteins that were highly connected in the network based on their functional properties. Ontology and functional enrichment analyses of these clusters revealed that the proteins in these clusters were represented in groups such as nucleotide-binding, kinase activity, GTPase activity, filamentous growth, MAPK signaling, and other signaling pathways. Twelve out of the 18 identified hub proteins were reestablished within these clusters suggesting that these proteins were the key players in hyphae development in Candida hyphae formation. Additionally, most of the proteins from the network were predicted to be localized on cell cortex or periphery of the fungi, which means they would act as the main protagonist in inducing infections within the host. Regulation of filamentous growth is the biological process that covered maximum number of protein nodes.Functional annotation analyzed by ClueGO provides a broad classification of these proteins and their involvement in various biological activities in addition to hyphal growth. Similarly, the ClueGO predicted three large clusters of proteins categorized on the basis of molecular function, biological process and cellular components. Within the molecular function, protein kinase activity is the largest one having 21 numbers of nodes and also kinase regulatory activity showed six nodes. Hence, the pathways related to kinase activity can be considered as one of the most important paths in C. albicans that is associated in integrating the environmental signals from a range of sources responsible for hyphae formation. Induction of hyphae is further transmitted through interaction of Cyr1 with Ras1 and Cap1 Cyr1 or Cdc35 is an essential enzyme of Candida. The interactomes of these above proteins were predicted to be involved in biological processes such as mitotic cell cycle process, cell morphogenesis, regulation of filamentous growth, intracellular signal transduction, and regulation of cellular component organization. Act1 was found to interact with many hyphae regulating proteins.Act1 is a hyphal tip associated protein and is required for hyphae elongation through hyphal tip polarization C. albicans. Lowering the Hog1 basal activity can promote Brg1 expression for hyphal elongation In our network, Hog1 and Hsp90 possessed the maximum number of connections and were believed to be hyphae suppressing proteins in Candida. Though its human homolog is present, it is considered as a drug target Candida that promotes the hyphae formation C. albicans infection Chk1 is needed for the survival in neutrophils and adherence to esophageal cells in human Among the hyphae forming proteins showing kinase activity, Hog1, Cek1 & Mkc1 proteins are the most essential kinases. In favorable condition hyphae formation takes place through Cek1 pathway. The activated Cek1 participates in the morphological transition through Cph1 hyphae specific transcription factor. The Cek1 protein is dephosphorylated by the phosphatase protein Cpp1. Cpp1 in turn is activated by Hog1 C. albicans. It is also to be noted that the hyphae formation is induced under different cellular conditions. However, in this case we considered only those data which were verified by wet-lab experiments. The data used in our study were generated considering the cellular conditions. Since the proposed model in this work was based on the experimentally verified data, the model took into account the different cellular conditions inherently.The proteins that showed multiple morphological features were Bcr1, Mkc1, Hwp1, Als1, Pmr1, Sod5, Cek1 and Pmt2. Most of them had direct interactions with the hyphae suppressing proteins such as Hog1, Hsp90 and Nrg1. Among these, Pmr1 is the most explored one, which shares 13 connections in the network, having positive role in hyphae formation and development. The other proteins require further attention to understand their role in hyphae formation in C. albicans, and their respective interactions necessary for the pathophysiology. From the study, it was concluded that the metabolic processes such as cAMP-mediated signaling, MAPK pathway and protein kinase pathway are significant for the morphogenesis and pathological activity of C. albicans. Proteins that are involved in both positive and negative regulation of hyphae formation are explored through network analyses and have also been described for their potential therapeutic targets. Further studies are in progress to elucidate the mechanisms for regulation of hyphae formation in C. albicans.Overall, this study emphasizes on the involvement of major hyphae forming proteins in different cellular physiology of Sanjib Das, Rajabrata Bhuyan, Angshuman Bagchi, Tanima Saha: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.DBT, Ministry of Science and Technology, Government of India, New Delhi. Angshuman Bagchi and Tanima Saha also received financial assistance from Personal Research Grant 2018–2019 provided by University of Kalyani.This work was supported by grants from BTIS net programme of The authors declare no conflict of interest.No additional information is available for this paper.