Network Pharmacological Analysis and Animal Experimental Study on Osteoporosis Treatment with GuBen-ZengGu Granules.

Aim: We explored the molecular pathway and material basis of GuBen-ZengGu granules (GBZGG) in treating osteoporosis using network pharmacology and animal experiments. Methods: The effective active components and potential targets of GBZGG were obtained from the TCMSP database and BATMAN-TCM database. Disease-related genes were obtained from GeneCard, NCBI, and DisGeNET. Next, a protein interaction network was established using the STRING database, and core genes were screened using the MCODE module. Cytoscape 3.8.0 was used to construct the network of component-disease-pathway-target, and KEGG pathway enrichment analyses were performed using the clusterProfiler R package to predict the mechanism of GBZGG in treating osteoporosis. An osteoporosis rat model was established by ovarian excision (OVX), and the partial results of network pharmacology were experimentally verified. Results: Pharmacodynamic results showed that GBZGG increased bone mineral density (BMD) and significantly improved the indexes of femur microstructure in model rats. The network pharmacology results showed that quercetin, luteolin, stigmasterol, angelicin, kaempferol, bakuchiol, bakuchiol, 7-O-methylisomucronulatum, isorhamnetin, formononetin, and beta-sitosterol are the major components of GBZGG, with MAPK1, AKT1, JUN, HSP90AA1, RELA, MAPK14, ESR1, RXRA, FOS, MAPK8, NCOA1, MYC, and IL-6 as its core targets for treating osteoporosis. Biological effects could be exerted by regulating the signaling pathways of fluid shear stress and the signaling pathways of atherosclerosis, advanced glycation end products (AGE-RAGE) of diabetic complications, prostate cancer, interleukin (IL-17), tumor necrosis factor (TNF), hepatitis B, mitogen-activated protein kinase (MAPK), etc. The results of animal experiments showed that GBZGG could reduce the serum levels of IL-6 and TNF-α, increase the expression of bone morphogenetic protein-2 (BMP-2) and runt-related transcription factor 2 (RUNX2) protein, and inhibit the activity of extracellular-regulated protein kinases (ERK1/2) and phosphorylation ERK1/2 (p-ERK1/2) protein. Conclusion: GBZGG reduces the expression of ERK1/2 and p-ERK1/2 proteins and mRNAs through the inhibitory effects on IL-6 and TNF-α and negatively regulates the MAPK/ERK signaling pathway. The osteoporosis model showed that it effectively improved the loss of bone mass and destruction of bone microstructure in rats and maintained a positive balance for bone metabolism.

[Human resources of community oral public health service and job satisfaction of the staff in Shanghai].

PURPOSE: To investigate the human resources of oral public health system and staff's job satisfaction in Shanghai, and to provide basis for improving the services of oral public health. METHODS: A census was performed among 246 health service centers in Shanghai from February to March 2019, and a questionnaire on oral public health staff's basic information of and job satisfaction was conducted. SPSS 20.0 software package was used for statistical analysis of job satisfaction and basic situation of employees. RESULTS: There were 759 community oral health service personnel in Shanghai, mainly composed of dentists (34.0%), public health physicians (21.96%), nurses (31.6%), and 24 general practitioners(3.2%) were also found. The profile of the staff was mainly 30-49 years old (69.8%), with bachelor degree (66.7%) and primary and intermediate professional titles (91.7%). Only 14.8% of them spend more than 24 hours in oral public health work every week. Community oral health workers had higher satisfaction with their superiors and colleagues, and lower satisfaction with their work income. Age, levels of professional title and years of oral health service were negatively correlated with job satisfaction. CONCLUSIONS: It is necessary to build a proper human resource system of community oral public health and improve the treatment of the staff, in order to promote the oral public health services in Shanghai.

[Survey on oral health knowledge, attitudes, behaviors among parents of disabled children in Shanghai area].

PURPOSE: To analyze the oral health behaviors of disabled children and their parents' oral health knowledge, attitudes in Shanghai city, and to provide information support for designing oral health care programs and making relevant policies. METHODS: By using stratified cluster sampling method, a questionnaire was given to 1381 parents of disabled children. The data were analyzed using SPSS 21.0 software package. RESULTS: The awareness rate of oral health knowledge among parents of disabled children was 67.21%, and 78.98% of parents had positive attitudes towards oral health. 13.61% of disabled children took sweet snacks before sleep, only 45.98% of disabled children brushed their teeth twice or more daily. 42.65% of disabled children used fluoride toothpaste, and 88.12% never flossed their teeth. The percentage of disabled children who had never visited a dentist was 49.75%. CONCLUSIONS: Oral health behaviors of disabled children in Shanghai city need to be improved, and the parents' oral health knowledge level is low. Customized educational programs should be carried out for parents, in order to strengthen oral health education in the suburban areas.

[Early childhood caries and its related risk factors in 1 296 children aged 3 to 5 years old in Shanghai].

PURPOSE: To investigate the prevelence of early childhood caries (ECC) and its related risk factors in children aged 3 to 5 years old in Shanghai. METHODS: According to the requirements of the Fourth National Epidemiological Survey of Oral Health, multistage, stratified, equal capacity and simple random sampling method was performed. A total of 1 296 children aged 3, 4 and 5 years old in 12 kindergartens in Shanghai were selected to calculate the prevalence and the mean dmft(decayed-missing-filled tooth). Their parents were surveyed with questionnaire on oral hygiene habit and consciousness of oral health and related factors. Statistical analysis were performed using SPSS 21.0 software package. RESULTS: The prevalence of ECC were 58.07% in children aged 3 to 5 years old in Shanghai, with dmft index of 2.99 in average. There were significant differences in the mean dmft and the prevalence between male and female and among different age groups(P<0.05). In multivariate logistic regression analysis, more frequent sweet drinks, eating sweets before sleep, less daily tooth brushing, starting tooth brushing after 2 years old and low level of parents' education and oral health knowledge were risk factors for dental caries. CONCLUSIONS: The prevalence of ECC in children aged 3 to 5 years old in Shanghai is severe. There is an urgent need for implementation of a systematic education on diet habit and oral health behavior, and the parental education on oral hygiene needs to be further improved.

Effect of Bacillus natto solid-state fermentation on the functional constituents and properties of Ginkgo seeds.

In the present investigation, fibrinolytic Ginkgo seeds were produced by solid-state fermentation (SSF) with Bacillus natto strains, and some parameters of the fermentation processes were investigated. Under optimal fermentation conditions, the fibrinolytic activity of Ginkgo seeds reached 3,682 ± 43 IU/g with the fermentation parameters of relative humidity 80%, initial water content 73%, fermentation temperature 38°C, inoculation volume 18%, and fermentation time 38 hr, respectively. The fermented Ginkgo seeds possessed a superior potential for the production of Nattokinase. What's more, the fermented Ginkgo seeds possessed higher total flavonoid and lower ginkgolic acids contents, which could enhance bioactivity and guarantee food safety. Sensory evaluations indicated that Ginkgo seeds produced by SSF could also be consumed as a kind of popular food. PRACTICAL APPLICATIONS: Fermented food is popular in countries. It can not only improve the sensory properties of the products, reduce undesirable constituents, and make nutrients easily absorbable, but also improve the nutritional properties. Ginkgo biloba L is one of the oldest species that has existed on earth for more than 200 million years. However, the application of Ginkgo seeds has been limited because of the ginkgolic acids. In a previous study, immobilized Bacillus natto acted upon Ginkgo seeds to enhance the bioactivity and safety of fermented Ginkgo seeds. However, separating the fermented Ginkgo seeds from the liquid needs a large amount of energy. The solid-state fermentation of Ginkgo seeds is a good choice to produce functional Ginkgo seed products.

Natural deep eutectic solvents couple with integrative extraction technique as an effective approach for mulberry anthocyanin extraction.

Natural deep eutectic solvents (NADES) have been growing interest as an alternative to the traditional organic solvents. They not only have the merit of high efficiency but also have the possibility to readily applicable to pharmaceutical and food applications. In the present study, NADES with high-speed homogenization and cavitation-burst extraction (HSH-CBE) was performed on fresh mulberry for anthocyanins extraction. The extraction conditions were statistically investigated by Plackett-Burman design (PBD) and Box-Behnken design (BBD). The optimal conditions were obtained as follows: chloride-citric acid-glucose formed a NADES with the mole ratio of 1:1:1, 30% water content, liquid-solid ratio 22 mL/g, homogenization time 60 s, homogenization speed 12,000 rpm, extraction time 30 min, negative pressure -0.08 MPa and extraction two times. The total maximum extraction of anthocyanins reached 6.05 mg/g fresh weight, which was 1.24 folds to those by the traditional organic solvents extraction. Moreover, NADES exhibited higher stability of anthocyanins extraction than traditional organic solvents, which was benefit for the analysis and preservation of anthocyanins. Consequently, this result revealed that the developed method could be taken as a sustainable, green and effective approach for anthocyanins extraction.

Application of integrative cloud point extraction and concentration for the analysis of polyphenols and alkaloids in mulberry leaves.

A simple and efficient method based on cloud point extraction and concentration combined with high performance liquid chromatography was developed for the simultaneous separation and determination of five target compounds (deoxynojirimycin, chlorogenic acid, rutin, isoquercitrin and astragalin) in mulberry leaves samples. Firstly, to obtain a high extraction rate, the ultrasound assisted extraction was developed on acid modified Triton X-114 system. Under the optimal conditions, the total maximum extraction yields of five target compounds reached 20.80 mg/g, which was superior to conventional solvent extraction. After the cloud point extraction and concentration, the HPLC analysis parameters of calibration curve, intra-day and inter-day precision, limits of detection for the five target compounds were determined systematically. The proposed method was successfully used to extract, concentrate, separate and determine the polyphenols and alkaloids in mulberry leaves.

[Clustering analysis in the pattern of KAP model on the application of oral health among students aged 12-15 in Shanghai].

PURPOSE: To assess the relation of oral health knowledge, attitude and practice(KAP) of the students aged 12-15 in Shanghai, and provide data support for intervention strategies of oral health. METHODS: A total of 2927 subjects aged 12-15 years old were selected according to the method of the Fourth National Oral Health Epidemiological Survey and the questionnaire was filled. SAS 9.4 software package was used for statistical analysis. RESULTS: The subjects were divided into 2 categories according to their knowledge and attitude of oral prevention and oral health. TypeⅠgroup, with high cognitive attitude, had more teeth brushing times than those of typeⅡgroup with poor cognitive attitudes(P<0.01). TypeⅠgroup had more people knowing and using toothpaste with fluoride. (P<0.01). The frequency of type I group using dental floss was significantly higher than typeⅡgroup(P<0.01), while the frequency of having deserts was relatively lower than typeⅡgroup (P<0.01), and the frequency of having drinks was lower than type Ⅱ(P<0.01). The two groups had no significant difference in the frequency of consumption of other drinks with sugar ( P=0.11). CONCLUSIONS: Oral related risk behaviors are quite common among 12-15 years students in Shanghai, which are closely related to the knowledge and attitude.

The relationship between apnoea hypopnoea index and Gensini score in patients with acute myocardial infarction undergoing emergency primary percutaneous coronary intervention.

BACKGROUND: Sleep apnoea hypopnoea syndrome (SAHS) is characterised by repetitive nocturnal hypoxemia and has a high prevalence among patients with acute myocardial infarction (AMI). But there are few studies on patients with AMI undergoing emergency primary percutaneous coronary intervention (pPCI). In this study, we want to find the prevalence of SAHS among patients with AMI undergoing emergency pPCI and determine whether SAHS would worsen the condition among these people, and especially affect the damage degree of the coronary artery. METHODS: Over four months, 95 patients admitted for the first time for AMI were observed. All of them had emergency primary PCIs. A total of 86 patients accepted the sleep study and were divided into four groups according to the apnoea hypopnoea index (AHI): SAHS was diagnosed when AHI ≥5/h and was defined as mild for AHI ≥5/h and <15/h, moderate for AHI ≥15/h and <30/h, and severe for AHI ≥30/h. On the contrary, the patients whose AHI <5/h were Non-SAHS. And the characteristics of the patients among these four groups were compared. According to the time of chest pain onset, the number of the patients between SAHS and non-SAHS, and patients' AHI during three intervals of one day were measured and compared; Makers including the sensitivity of serum troponin T (hs-TnT), creatine kinase isoenzyme MB (CK-MB), left ventricular ejection fraction (LVEF), pro-brain-type natriuretic peptide (pro-BNP), Gensini score and collateral vessels between the SAHS and non-SAHS were compared. And the relationships between the AHI of these patients and the markers were analysed. RESULTS: Of the 86 patients studied, 65 had SAHS, representing a SAHS prevalence of 75.58% among patients with AMI undergoing emergency pPCI. There were significant differences in average ages, smoking and arrhythmia (P<0.05) between these four groups. There was no significant difference between AMI patients with or without SAHS regarding the day-night pattern. But there showed significant differences between SAHS and non-SAHS in Gensini score (P<0.05) and pro-BNP (P<0.05). Also, there were positive correlations between AHI and Gensini score (r=0.490, P<0.05) and pro-BNP (r=0.338, P<0.05). CONCLUSIONS: Among patients with AMI undergoing emergency pPCI, there is a high prevalence of SAHS. There are also positive correlations between AHI and Gensini score, and pro-BNP. Therefore, guided by the results, should we conduct a routine screening to those patients normally and could we relieve the damage to the coronary artery by curing the SAHS?

Easy and Fast Western Blotting by Thin-Film Direct Coating with Suction.

Thin-film direct coating (TDC) has been successfully used in Western blotting (WB). In this study, the advanced technique of TDC with suction (TDCS) was developed to reduce the consumption amount of antibody by a factor of up to 10(4) in comparison with the amount consumed by the conventional WB using the capillary tube without any need of special micromachining processes. The operation time for completely finishing a high-quality WB can be reduced from 3 h in conventional WB to about 5 min or even less by TDCS. In addition, the signal-to-noise ratio of the immunoblotting by TDCS can be markedly increased. TDCS WB showed a high linearity within a 6-log2 dynamic range for detecting 90-6000 ng of purified recombinant glutathione-S-transferase (GST) proteins and could particularly detect extrinsic GST proteins added in crude Escherichia coli or 293T cell lysates. Moreover, a protein mixture containing bovine serum albumin, GST, and ubiquitin could be specifically probed in parallel with their corresponding antibodies through multichannel TDCS WB. This simple and innovative TDCS WB offers various potential applications in simultaneously finishing multiple antibody-antigen screenings in a fast and single experiment.

New Is Old, and Old Is New: Recent Advances in Antibiotic-Based, Antibiotic-Free and Ethnomedical Treatments against Methicillin-Resistant Staphylococcus aureus Wound Infections.

Staphylococcus aureus is the most common pathogen of wound infections. Thus far, methicillin-resistant S. aureus (MRSA) has become the major causative agent in wound infections, especially for nosocomial infections. MRSA infections are seldom eradicated by routine antimicrobial therapies. More concerning, some strains have become resistant to the newest antibiotics of last resort. Furthermore, horizontal transfer of a polymyxin resistance gene, mcr-1, has been identified in Enterobacteriaceae, by which resistance to the last group of antibiotics will likely spread rapidly. The worst-case scenario, "a return to the pre-antibiotic era", is likely in sight. A perpetual goal for antibiotic research is the discovery of an antibiotic that lacks resistance potential, such as the recent discovery of teixobactin. However, when considering the issue from an ecological and evolutionary standpoint, it is evident that it is insufficient to solve the antibiotic dilemma through the use of antibiotics themselves. In this review, we summarized recent advances in antibiotic-based, antibiotic-free and ethnomedical treatments against MRSA wound infections to identify new clues to solve the antibiotic dilemma. One potential solution is to use ethnomedical drugs topically. Some ethnomedical drugs have been demonstrated to be effective antimicrobials against MRSA. A decline in antibiotic resistance can therefore be expected, as has been demonstrated when antibiotic-free treatments were used to limit the use of antibiotics. It is also anticipated that these drugs will have low resistance potential, although there is only minimal evidence to support this claim to date. More clinical trials and animal tests should be conducted on this topic.

Western blotting by thin-film direct coating.

A novel thin-film direct coating (TDC) technique was developed to markedly reduce the amount of antibody required for Western blotting (WB). Automatic application of the technique for a few seconds easily and homogeneously coats the specific primary antibody on the polyvinylidene fluoride (PVDF) membrane. While conventional WB requires 0.4 µg of the primary antibody, the proposed technique only uses 4 × 10(-2) µg, which can be reduced further to 4 × 10(-5) µg by reducing the coater width. Moreover, the proposed process reduces antibody probing times from 60 to 10 min. The quantification capability of TDC WB showed high linearity within a 4-log2 dynamic range for detecting target antigen glutathione-S-transferase. Furthermore, TDC WB can specifically detect the extrinsic glutathione-S-transferase added in the Escherichia coli or 293T cell lysate with better staining sensitivity than conventional WB. TDC WB can also clearly probe the intrinsic ß-actin, α-tubulin, and glyceraldehyde 3-phosphate dehydrogenase, which are usually used as control proteins in biological experiments. This novel technique has been shown to not only have valuable potential for increasing WB efficiency but also for providing significant material savings for future biomedical applications.

BT cationic peptides: small peptides that modulate innate immune responses of chicken heterophils and monocytes.

Neonatal poultry exhibit a transient susceptibility to infectious diseases during the first week of life that stems from inefficient host defense mechanisms. Yet, the initial host immune response to pathogens is a critical determinant of disease resistance and susceptibility. With this context in mind, novel ways to stimulate or modulate the hosts' natural immune response is emerging as an important area of interest for food animal producers including the poultry industry. Specifically, we have been investigating new modulation strategies tailored around the selective stimulation of the host's immune system, and particularly rapid acting innate immunity, as an alternative to direct targeting of microbial pathogens. One such approach that we have been investigating is the use of a group of cationic peptides produced by a Gram-positive soil bacterium, Brevibacillus texasporus (BT peptides). We have previously shown that, provided as a feed additive, BT peptides significantly induced a concentration-dependent protection against cecal colonization and extraintestinal colonization by Salmonella enterica serovar Enteritidis (SE). This protection is not the result of direct antibacterial activity of the BT peptides on the SE since the concentrations used were below the minimum inhibitory concentration for SE. We also found that BT are not absorbed in the intestine, but still induce a significant up-regulation in the functional efficiency of peripheral blood heterophils and monocytes. The mechanisms of this immune modulation are unknown. Here, using in vitro models for measuring: (1) leukocyte oxidative burst, (2) changes in leukocyte cytokine and chemokines gene expression profiles, and (3) phosphorylation of the mitogen activated protein kinases (MAPKs) in leukocytes, we evaluated the role of BT peptides as priming mediators for heterophil and monocyte responses at the level of cell function, gene transcription/expression, and cell phosphorylation following stimulation with inflammatory agonists. BT peptides primed both heterophils and monocytes for an increased oxidative burst and up-regulation in transcription of the pro-inflammatory cytokines IL-1ß and IL-6 and inflammatory chemokines CXCLi1 and CXCLi2 induced by inflammatory agonists. In addition, BT peptides induced a rapid (10min) phosphorylation and activation of the extracellular signal-regulated kinase (ERK1/2) and p38 kinase pathways in primary chicken heterophils. Taken together, we conclude that BT peptides, acting through MAPK pathways, enhance leukocyte functional and pro-inflammatory cytokine and chemokine gene transcription activities. These small cationic peptides may prove useful as immune modulators in neonatal poultry.

An essential role of Tap42-associated PP2A and 2A-like phosphatases in Ty1 transcriptional silencing of S. cerevisiae.

We previously reported that overproduction of non-translatable mRNA silences Ty1 transcription, possibly via functional inactivation of the nuclear cap-binding complex (CBC) and subsequent hyperstimulation of the TORC1 pathway. Further experimental evidence for CBC-to-TORC1 signalling in Ty1 transcriptional silencing is presented here. The role of Tap42 (a key downstream component of the TORC1 pathway) was tested. Mutations affecting components of the Tap42-associated PP2A/2A-like phosphatases (Tap42, redundant Pph21/Pph22 and Sit4) eliminate Ty1 transcriptional silencing and epistasis experiments show that the phosphatases function downstream of CBC. Thus, Tap42 functions in the same positive direction as Sit4, Pph21 and Pph22 in Ty1 transcriptional silencing, providing support to the idea that Tap42 may play a positive role in phosphatase activity in response to TORC1 signalling. Moreover, the pph21-102 and sit4-102 mutations affecting interactions of the phosphatases with the Tap42 regulator also abolish Ty1 transcriptional silencing, confirming that the Tap42-phosphatase complex is one required for TORC1-mediated Ty1 transcriptional silencing. PP2A holoenzyme activity, which is independent of Tap42 and TORC1, is not essential for Ty1 transcriptional silencing, strengthening the argument that TORC1-specific signalling underlies Ty1 transcriptional silencing.

Overproduction of non-translatable mRNA silences. The transcription of Ty1 retrotransposons in S. cerevisiae via functional inactivation of the nuclear cap-binding complex and subsequent hyperstimulation of the TORC1 pathway.

Co-suppression is high gene copy number-triggered homology-dependent gene silencing, and co-suppression may have evolved in eukaryotes to counter invasive molecular parasites, such as viruses and transposons. We previously reported 'Ty1 transcriptional co-suppression'-high Ty1 copy number-triggered transient transcriptional silencing of Ty1 retrotransposons in S. cerevisiae. We report here that this phenomenon is unlikely to be homology-dependent, despite the copy number dependence. The Ty1 mRNA is an extremely poor template for translation, and overproduction of non-translatable mRNA without Ty1 homology is sufficient to initiate the transient Ty1 transcriptional silencing. We present genetic evidence that overproduction of non-translatable mRNA may functionally inactivate the nuclear cap-binding complex (CBC), and inactivation of CBC may then hyperstimulate the TORC1 pathway to mediate Ty1 transcriptional silencing. Our results point to a potent regulatory function of non-translatable mRNA in vivo (via CBC and TORC1) to potentially modulate a variety of intracellular activities, such as Ty1 transcription. Although overproduction of non-translatable mRNA causes transient Ty1 transcriptional silencing, it does not play a detectable role in controlling Ty1 retrotransposition.

Structure and biosynthesis of the BT peptide antibiotic from Brevibacillus texasporus.

We isolated a novel gram-positive bacterium, Brevibacillus texasporus, that produces an antibiotic, BT. BT is a group of related peptides that are produced by B. texasporus cells in response to nutrient limitation. We report here purification and determination of the structure of the most abundant BT isomer, BT1583. Amino acid composition and tandem mass spectrometry experiments yielded a partial BT1583 structure. The presence of ornithine and d-form residues in the partial BT1583 structure indicated that the peptide is synthesized by a nonribosomal peptide synthetase (NRPS). The BT NRPS operon was rapidly and accurately identified by using a novel in silico NRPS operon hunting strategy that involved direct shotgun genomic sequencing rather than the unreliable cosmid library hybridization scheme. Sequence analysis of the BT NRPS operon indicated that it encodes a colinear modular NRPS with a strict correlation between the NRPS modules and the amino acid residues in the peptide. The colinear nature of the BT NRPS enabled us to utilize the genomic information to refine the BT1583 peptide sequence to Me(2)-4-methyl-4-[(E)-2-butenyl]-4,N-methyl-threonine-L-dO-I-V-V-dK-V-dL-K-dY-L-V-CH2OH. In addition, we report the discovery of novel NRPS codons (sets of the substrate specificity-conferring residues in NRPS modules) for valine, lysine, ornithine, and tyrosine.

Possible integration of upstream signals at Cdc42 in filamentous differentiation of S. cerevisiae.

Various environmental stimuli (such as nitrogen starvation, short-chain alcohols and slowed DNA synthesis) induce filamentous differentiation in S. cerevisiae. Genetic mutations (such as deletion of the mitotic cyclin gene CLB2) cause constitutive filamentous differentiation. Although different stimulus-induced filamentous differentiation involves different signalling pathways, Cdc42 has been identified as a common regulator. We show here that Cdc42 is also required for hydroxyurea (HU)-induced and clb2Delta-caused filamentous growth. We show that the mitotic CDK Clb2/Cdc28 functions upstream of Cdc42 in regulating filamentous differentiation. This result points to possible existence of a Cdc42-MAPK-Clb2/Cdc28 positive feedback loop in the signalling of filamentous differentiation. We report isolation of a cdc42-Y40F allele that blocks HU-induced, but not nitrogen starvation-induced, short-chain alcohol-induced or clb2Delta-caused, filamentation. Based on these results, we propose a model in which Cdc42 functions as a possible integrator for the upstream signals of filamentous differentiation (from the filamentous growth MAPK pathway, the cAMP pathway and the Mec1/Rad53 checkpoint pathway). We also show evidence that the mitotic CDK inhibitor Swe1 may mediate the cross-talk between the cAMP and MAPK pathways.

Genetic/genomic evidence for a key role of polarized endocytosis in filamentous differentiation of S. cerevisiae.

Unicellular S. cerevisiae cells switch from the yeast form to pseudohyphal or filamentous form in response to environmental cues. We report that wild-type BY diploids (in which yeast ORFs have been systematically deleted) undergo normal HU-induced filamentous growth and discernable nitrogen starvation-induced filamentous growth, despite their perceived filamentation-deficient S288C genetic background. This finding allowed us to perform a genome-wide survey for non-essential genes that are required for filamentous growth with the homozygous deletion strains. We report that genes involved in endocytosis are required for both HU-induced and nitrogen starvation-induced filamentous growth. Surprisingly, no known genes involved in exocytosis are required. Despite the fact that polarized growth involves transport of vesicles to the site of growth, we failed to obtain genetic/genomic evidence that exocytosis plays an essential role in filamentous growth. A possible key role of polarized endocytosis (from the growth tip) is consistent with the proposed biological function of filamentous growth as a foraging behaviour. In addition, BUD8 that encodes the distal landmark in yeast-form bipolar budding is required for nitrogen starvation-induced but not HU-induced filamentous growth. Moreover, BUD5, SPA2, PEA2 and BUD6 that regulate bipolar bud site selection do not regulate the unipolar distal budding pattern in HU-induced filamentous growth.

Genome-wide survey of non-essential genes required for slowed DNA synthesis-induced filamentous growth in yeast.

We recently discovered that slowed DNA synthesis induces filamentous differentiation in S. cerevisiae. We screened the BY yeast deletion strains and identified four classes of non-essential genes that are required for both slowed DNA-induced filamentous growth and classic forms of filamentous growth: (a) genes encoding regulators of the actin cytoskeleton and cell polarity, ABP1, CAP2 and HUF1 (=YOR300W), in addition to the previously known BNI1, BUD2, PEA2, SPA2 and TPM1; (b) genes that are likely involved in cell wall biosynthesis, ECM25, GAS1 and PRS3; (c) genes encoding possible regulators of protein secretion, SEC66, RPL21A and RPL34B; (d) genes encoding factors for normal mitochondrial function, IML1 and UGO1. These results showed that pseudohyphal formation involves not the only previously known regulation of the actin cytoskeleton/cell polarity but also regulation of cell wall synthesis, protein secretion and mitochondrial function. Identification of multiple classes of genes that are required for both slowed DNA synthesis-induced and classic forms of filamentous growth confirms that slowed DNA synthesis-induced filamentous growth is bone fide filamentous differentiation.

Induction of S. cerevisiae filamentous differentiation by slowed DNA synthesis involves Mec1, Rad53 and Swe1 checkpoint proteins.

A key question in eukaryotic differentiation is whether there are common regulators or biochemical events that are required for diverse types of differentiation or whether there is a core mechanism for differentiation. The unicellular model organism Saccharomyces cerevisiae undergoes filamentous differentiation in response to environmental cues. Because conserved cell cycle regulators, the mitotic cyclin-dependent kinase Clb2/Cdc28, and its inhibitor Swe1 were found to be involved in both nitrogen starvation- and short chain alcohol-induced filamentous differentiation, they were identified as components of the core mechanism for filamentous differentiation. We report here that slowed DNA synthesis also induces yeast filamentous differentiation through conserved checkpoint proteins Mec1 and Rad53. Swe1 and Clb2 are also involved in this form of differentiation, and the core status of Swe1/Clb2/Cdc28 in the mechanism of filamentous differentiation has therefore been confirmed. Because the cAMP and filamentous growth mitogen-activated protein kinase pathways that mediate nitrogen starvation-induced filamentous differentiation are not required for slowed DNA synthesis-induced filamentous growth, they can therefore be excluded from the core mechanism. More significantly, slowed DNA synthesis also induces differentiation in mammalian cancer cells, and such stimulus conservation may indicate that the core mechanism for yeast filamentous differentiation is conserved in mammalian differentiation.