Beyond energy provider: multifunction of lipid droplets in embryonic development.

Since the discovery, lipid droplets (LDs) have been recognized to be sites of cellular energy reserves, providing energy when necessary to sustain cellular life activities. Many studies have reported large numbers of LDs in eggs and early embryos from insects to mammals. The questions of how LDs are formed, what role they play, and what their significance is for embryonic development have been attracting the attention of researchers. Studies in recent years have revealed that in addition to providing energy for embryonic development, LDs in eggs and embryos also function to resist lipotoxicity, resist oxidative stress, inhibit bacterial infection, and provide lipid and membrane components for embryonic development. Removal of LDs from fertilized eggs or early embryos artificially leads to embryonic developmental arrest and defects. This paper reviews recent studies to explain the role and effect mechanisms of LDs in the embryonic development of several species and the genes involved in the regulation. The review contributes to understanding the embryonic development mechanism and provides new insight for the diagnosis and treatment of diseases related to embryonic developmental abnormalities.

Insights into the reduction of antibiotic-resistant bacteria and mobile antibiotic resistance genes by black soldier fly larvae in chicken manure.

The increasing prevalence of antibiotic-resistant bacteria (ARB) from animal manure has raised concerns about the potential threats to public health. The bioconversion of animal manure with insect larvae, such as the black soldier fly larvae (BSFL, Hermetia illucens [L.]), is a promising technology for quickly attenuating ARB while also recycling waste. In this study, we investigated BSFL conversion systems for chicken manure. Using metagenomic analysis, we tracked ARB and evaluated the resistome dissemination risk by investigating the co-occurrence of antibiotic resistance genes (ARGs), mobile genetic elements (MGEs), and bacterial taxa in a genetic context. Our results indicated that BSFL treatment effectively mitigated the relative abundance of ARB, ARGs, and MGEs by 34.9%, 53.3%, and 37.9%, respectively, within 28 days. Notably, the transferable ARGs decreased by 30.9%, indicating that BSFL treatment could mitigate the likelihood of ARG horizontal transfer and thus reduce the risk of ARB occurrence. In addition, the significantly positive correlation links between antimicrobial concentration and relative abundance of ARB reduced by 44.4%. Moreover, using variance partition analysis (VPA), we identified other bacteria as the most important factor influencing ARB, explaining 20.6% of the ARB patterns. Further analysis suggested that antagonism of other bacteria on ARB increased by 1.4 times, while nutrient competition on both total nitrogen and crude fat increased by 2.8 times. Overall, these findings provide insight into the mechanistic understanding of ARB reduction during BSFL treatment of chicken manure and provide a strategy for rapidly mitigating ARB in animal manure.

Ammonia-induced excess ROS causes impairment and apoptosis in porcine IPEC-J2 intestinal epithelial cells.

Ammonia is one of the most important toxic metabolites in the intestine of animals. It can cause intestinal damage and associated intestinal diseases through different endogenous or exogenous stimuli. However, the definition of harmful ammonia concentration and the molecular mechanism of ammonia - induced intestinal epithelial injury remain unclear. In this study, we found that the viability of porcine IPEC-J2 intestinal epithelial cells significantly decreased with the increase of NH4Cl dose (20-80 mM). Ammonia (40 mM NH4Cl) increased the expression level of ammonia transporter RHCG and disrupted the intestinal barrier function of IPEC-J2 cells by reducing the expression levels of the tight junction molecules ZO-1 and Claudin-1. Ammonia caused elevated levels of ROS and apoptosis in IPEC-J2 cells. This was manifested by decreased activity of antioxidant enzymes SOD and GPx, decreased mitochondrial membrane potential, and increased cytoplasmic Ca2+ concentration. In addition, the expression levels of apoptosis-related molecules Caspase-9, Caspase-3, Fas, Caspase-8, p53 and Bax were increased, the expression level of anti-apoptotic molecule Bcl-2 was decreased. Moreover, the antioxidant NAC (N-acetyl-L-cysteamine) effectively alleviated ammonia-induced cytotoxicity, reduced ROS level, Ca2+ concentration, and the apoptosis of IPEC-J2 cells. The results suggest that ammonia-induced excess ROS triggered apoptosis through mitochondrial pathway, death receptor pathway and DNA damage. This study can provide reference and theoretical basis for the definition of harmful ammonia concentration in pig intestine and the effect and mechanism of ammonia on pig intestinal health.

ARP2/3 Regulates Fatty Acid Synthesis by Modulating Lipid Droplets' Motility.

The breakdown of lipid droplets (LDs) provides energy and contributes to the proliferation and migration of cancer cells. Recent studies have suggested that motility plays a key role in LD breakdown. However, the molecular mechanisms underlying LD motility were poorly characterized. In this study, we examined the function of microfilament-associated proteins 2 and 3 (ARP2 and ARP3) in regulating LDs' motility in Hela cells. ARP2/3 mediated the LDs' physical contact with F-actin and promoted the recruitment of Myosin Heavy Chain 9 (MYH9). MYH9 regulated the LD content by binding with LDs and ARP2/3. The number of LDs and TG content was increased after MYH9 interfered. The genes related to FA-related genes and neutral lipid synthesis-related genes were significantly increased (p < 0.05) when ARP2 and ARP3 were overexpressed. Bioinformatic analysis indicated that the high expression of ARP2/3 was associated with a poorer prognosis in cervical squamous cell carcinoma (CSCC). This study showed the effect of cytoskeletal filaments on LD metabolism in cancer cells.

The RNA surveillance factor UPF1 regulates the migration and adhesion of porcine skeletal muscle satellite cells.

Skeletal muscle satellite cells (SCs) play an important role in the repairment and regeneration of damaged muscle. The activation, proliferation, migration, and differentiation of SCs are essential to the response to muscle injury. Up-frameshift 1 (UPF1) is involved in the regulation of many developmental processes. However, the role of UPF1 and its associated regulatory mechanism in SCs are still unclear. Here, we analyzed changes in the transcriptome of porcine SCs with UPF1 knockdown. The results showed that focal adhesion and actin cytoskeleton processes were regulated by UPF1. We also confirmed experimentally that UPF1 promoted SC migration and adhesion by regulating the expression of F-Actin, Vinculin, and several adhesion-related genes. Furthermore, we found that phosphorylated focal adhesion kinase (p-FAK) was down-regulated by UPF1 knockdown. This study identifies the role of UPF1 in regulating SC migration and adhesion and therefore provides new insight into the regulatory mechanism of UPF1 in the process of repairing damaged muscle.

Motility Plays an Important Role in the Lifetime of Mammalian Lipid Droplets.

The lipid droplet is a kind of organelle that stores neutral lipids in cells. Recent studies have found that in addition to energy storage, lipid droplets also play an important role in biological processes such as resistance to stress, immunity, cell proliferation, apoptosis, and signal transduction. Lipid droplets are formed at the endoplasmic reticulum, and mature lipid droplets participate in various cellular processes. Lipid droplets are decomposed by lipase and lysosomes. In the life of a lipid droplet, the most important thing is to interact with other organelles, including the endoplasmic reticulum, mitochondria, peroxisomes, and autophagic lysosomes. The interaction between lipid droplets and other organelles requires them to be close to each other, which inevitably involves the motility of lipid droplets. In fact, through many microscopic observation techniques, researchers have discovered that lipid droplets are highly dynamic organelles that move quickly. This paper reviews the process of lipid droplet motility, focusing on explaining the molecular basis of lipid droplet motility, the factors that regulate lipid droplet motility, and the influence of motility on the formation and decomposition of lipid droplets. In addition, this paper also proposes several unresolved problems for lipid droplet motility. Finally, this paper makes predictions about the future research of lipid droplet motility.

Faster and More Specific: Excited-State Intramolecular Proton Transfer-Based Dyes for High-Fidelity Dynamic Imaging of Lipid Droplets within Cells and Tissues.

Lipid droplets (LDs), a type of dynamic organelle residing at the center of cellular lipid storage, have been identified to play important roles in multiple biological processes, metabolic disorders, and diseases. The highly dynamic characters of LDs were found to correspond to their physiological and pathological functions. Hence, the fluorescent probes which enable dynamic tracking of LDs should be very helpful for better understanding the mechanisms of LDs involved biological processes and diseases. Herein we present, to the best of our knowledge, the first class of excited-state intramolecular proton transfer (ESIPT) fluorescence dyes (Flp-(11-13, 19)) for dynamic imaging of LDs based on 3-hydroxyflavone (3HF) derivatives. Flp-(11-13, 19) display strong fluorescence from yellow to NIR in lipid but exhibit almost nonfluorescence in aqueous solution. Besides, they also show large Stokes shifts (>150 nm), narrow absorption and emission peaks, and good oil-water separation efficiency, which makes them specifically target and stain LDs with very low background noisy in both living cells and fixed cells. They stain intracellular LDs quite quickly (within 30 s) with very low dosage (as low as 500 nM). Benefitting from these advantages, Flp-(11-13, 19) are applied successfully in tracking the dynamic nature of LDs and accumulation of LDs in both aqueous solution and living cells, 3D imaging of LDs for visualization of their repartition within the cells, and visualizing LDs in tissues of diseases mice models including adipose, skeletal muscle, and fatty liver tissues, underscoring the potential utility of these dyes in both LDs biology research and medical diagnosis of LDs involved diseases.

SEIPIN: A Key Factor for Nuclear Lipid Droplet Generation and Lipid Homeostasis.

Lipid homeostasis is essential for normal cell physiology. Generally, lipids are stored in a lipid droplet (LD), a ubiquitous organelle consisting of a neutral lipid core and a single layer of phospholipid membrane. It is thought that LDs are generated from the endoplasmic reticulum and then released into the cytosol. Recent studies indicate that LDs can exist in the nucleus, where they play an important role in the maintenance of cell phospholipid homeostasis. However, the details of nuclear lipid droplet (nLD) generation have not yet been clearly characterized. SEIPIN is a nonenzymatic protein encoded by the Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2) gene. It is associated with lipodystrophy diseases. Many recent studies have indicated that SEIPIN is essential for LDs generation. Here, we review much of this research in an attempt to explain the role of SEIPIN in nLD generation. From an integrative perspective, we conclude by proposing a theoretical model to explain how SEIPIN might participate in maintaining homeostasis of lipid metabolism.

PSMD1 and PSMD2 regulate HepG2 cell proliferation and apoptosis via modulating cellular lipid droplet metabolism.

BACKGROUND: Obesity and nonalcoholic steatohepatitis (NASH) are well-known risk factors of hepatocellular carcinoma (HCC). The lipid-rich environment enhances the proliferation and metastasis abilities of tumor cells. Previous studies showed the effect of the ubiquitin-proteasome system (UPS) on tumor cell proliferation. However, the underlying mechanism of UPS in regulating the proliferation of lipid-rich tumor cells is not totally clear. RESULTS: Here, we identify two proteasome 26S subunits, non-ATPase 1 and 2 (PSMD1 and PSMD2), which regulate HepG2 cells proliferation via modulating cellular lipid metabolism. Briefly, the knockdown of PSMD1 and/or PSMD2 decreases the formation of cellular lipid droplets, the provider of the energy and membrane components for tumor cell proliferation. Mechanically, PSMD1 and PSMD2 regulate the expression of genes related to de novo lipid synthesis via p38-JNK and AKT signaling. Moreover, the high expression of PSMD1 and PSMD2 is significantly correlated with poor prognosis of HCC. CONCLUSION: We demonstrate that PSMD1 and PSMD2 promote the proliferation of HepG2 cells via facilitating cellular lipid droplet accumulation. This study provides a potential therapeutic strategy for the treatment of lipid-rich tumors.

Reactive Oxygen Species Induces Lipid Droplet Accumulation in HepG2 Cells by Increasing Perilipin 2 Expression.

Non-alcoholic fatty liver disease (NAFLD) has become the world's most common liver disease. The disease can develop liver fibrosis or even carcinomas from the initial hepatic steatosis, and this process is influenced by many factors. Reactive oxygen species (ROS), as potent oxidants in cells, have been reported previously to play an important role in the development of NAFLD progression via promoting neutral lipid accumulation. Here, we found that ROS can promote lipid droplet formation in hepatocytes by promoting perilipin2 (PLIN2) expression. First, we used different concentrations of hydrogen peroxide to treat HepG2 cells and found that the number of lipid droplets in the cells increased, however also that this effect was dose-independent. Then, the mRNA level of several lipid droplet-associated genes was detected with hydrogen peroxide treatment and the expression of PLIN2, PLIN5, and FSP27 genes was significantly up-regulated (p < 0.05). We overexpressed PLIN2 in HepG2 cells and found that the lipid droplets in the cells were markedly increased. Interference with PLIN2 inhibits ROS-induced lipid droplet formation, revealing that PLIN2 is a critical factor in this process. We subsequently analyzed the regulatory pathway and protein interaction network that is involved in PLIN2 and found that PLIN2 can regulate intracellular lipid metabolism through the PPARα/RXRA and CREB/CREBBP signaling pathways. The majority of the data indicated the correlation between hydrogen peroxide-induced PLIN2 and lipid droplet upregulation. In conclusion, ROS up-regulates the expression of PLIN2 in hepatocytes, whereas PLIN2 promotes the formation of lipid droplets resulting in lipid accumulation in liver tissues.

MicroRNA 433 regulates nonsense-mediated mRNA decay by targeting SMG5 mRNA.

BACKGROUND: Nonsense-mediated mRNA decay (NMD) is a RNA quality surveillance system for eukaryotes. It prevents cells from generating deleterious truncated proteins by degrading abnormal mRNAs that harbor premature termination codon (PTC). However, little is known about the molecular regulation mechanism underlying the inhibition of NMD by microRNAs. RESULTS: The present study demonstrated that miR-433 was involved in NMD pathway via negatively regulating SMG5. We provided evidence that (1) overexpression of miR-433 significantly suppressed the expression of SMG5 (P < 0.05); (2) Both mRNA and protein expression levels of TBL2 and GADD45B, substrates of NMD, were increased when SMG5 was suppressed by siRNA; (3) Expression of SMG5, TBL2 and GADD45B were significantly increased by miR-433 inhibitor (P < 0.05). These results together illustrated that miR-433 regulated NMD by targeting SMG5 mRNA. CONCLUSIONS: Our study highlights that miR-433 represses nonsense mediated mRNA decay. The miR-433 targets 3'-UTR of SMG5 and represses the expression of SMG5, whereas NMD activity is decreased when SMG5 is decreased. This discovery provides evidence for microRNA/NMD regulatory mechanism.

Six1: a critical transcription factor in tumorigenesis.

In the past two decades, many studies have shown that sine oculis homeobox 1 (Six1) is a powerful regulator of organogenesis and disease, with important roles in tumorigenesis; therefore, it is important to review the biology of Six1 gene comprehensively. This review describes the function of Six1 in normal organ development, summarizes its role in several diseases, including cancer. The review will extend our understanding about the functional roles of Six1 and suggests opportunities to target Six1 for diagnostic, prognostic, and therapeutic purposes.

MyoD Is a Novel Activator of Porcine FIT1 Gene by Interacting with the Canonical E-Box Element during Myogenesis.

Fat-induced transcript 1 (FIT1/FITM1) gene is a member of the conserved gene family important for triglyceride-rich lipid droplet accumulation. FIT1 gene displays a similar muscle-specific expression across pigs, mice, and humans. Thus pigs can act as a useful model of many human diseases resulting from misexpression of FIT1 gene. Triglyceride content in skeletal muscle plays a key role in pork meat quality and flavors. An insertion/deletion mutation in porcine FIT1 coding region shows a high correlation with a series of fat traits. To gain better knowledge of the potential role of FIT1 gene in human diseases and the correlations with pork meat quality, our attention is given to the region upstream of the porcine FIT1 coding sequence. We cloned ~1 kb of the 5'-flanking region of porcine FIT1 gene to define the role of this sequence in modulating the myogenic expression. A canonical E-box element that activated porcine FIT1 promoter activity during myogenesis was identified. Further analysis demonstrated that promoter activity was induced by overexpression of MyoD1, which bound to this canonical E-box during C2C12 differentiation. This is the first evidence that FIT1 as the direct novel target of MyoD is involved in muscle development.

The formation of brown adipose tissue induced by transgenic over-expression of PPARγ2.

Brown adipose tissue (BAT) is specialized to dissipate energy as heat, therefore reducing fat deposition and counteracting obesity. Brown adipocytes arise from myoblastic progenitors during embryonic development by the action of transcription regulator PRDM16 binding to PPARγ, which promotes BAT-like phenotype in white adipose tissue. To investigate the capability of converting white adipose tissue to BAT or browning by PPARγ in vivo, we generated transgenic mice with over-expressed PPARγ2. The transgenic mice showed strong brown fat features in subcutaneous fat in morphology and histology. To provide molecular evidences on browning characteristics of the adipose tissue, we employed quantitative real-time PCR to determine BAT-specific gene expressions. The transgenic mice had remarkably elevated mRNA level of UCP1, Elovl3, PGC1α and Cebpα in subcutaneous fat. Compared with wild-type mice, UCP1 protein levels were increased significantly in transgenic mice. ATP concentration was slightly decreased in the subcutaneous fat of transgenic mice. Western blotting analysis also confirmed that phosphorylated AMPK and ACC proteins were significantly (P<0.01) increased in the transgenic mice. Therefore, this study demonstrated that over-expression of PPARγ2 in skeletal muscle can promote conversion of subcutaneous fat to brown fat formation, which can have beneficial effects on increasing energy metabolisms and combating obesity.

Regulation of nitrogen transformation and microbial community by inoculation during livestock manure composting.

This study examined the effects of three Bacillus strains and one Saccharomyces cerevisiae strain on nitrogen transformation and microbial communities in pig and chicken manure compost. The findings revealed that the use of compound microbial inoculants increased the compost temperature, accelerated moisture reduction, enhanced cellulase activity, and stimulated the accumulation of NH4 +-N, NO3 --N, and total nitrogen (TN), resulting in a 9% increase in TN content. The abundance of Firmicutes decreased by 3.95% at the maturation phase, while Actinobacteria and Bacteroidetes increased by 1.64% and 1.85%, respectively. Inoculation led to an increase in amoA, nxrA and nifH gene copy numbers, while simultaneously reducing the abundance of nirK, nosZ and nirS genes. It also resulted in an increase in functional enzyme levels, specifically nif and amo, with a corresponding decrease in nor. Clostridium, Phascolarctobacterium, Eubacterium and Faecalibacterium from the class Clostridium, which have a significant correlation with nifH and nxrA genes, suggest their likely crucial role in nitrogen retention and fixation. Inoculation aided in the removal of pathogenic bacteria and antibiotic resistance genes (ARGs) like fluoroquinolones, nucleosides and nitroimidazole. This study provides effective theoretical support for the mechanism of nitrogen retention and fixation, and for improving the quality of compost.

Intervention effects of fructooligosaccharide and astragalus polysaccharide, as typical antibiotic alternatives, on antibiotic resistance genes in feces of layer breeding: advantages and defects.

Although antibiotic alternatives are widely used in livestock and poultry breeding industry after in-feed antibiotics ban, their intervention effects on antibiotic resistance genes (ARGs) in these food animals' feces remain poorly understood. Here effects of fructooligosaccharide (FOS) and astragalus polysaccharide (APS), as typical antibiotic alternatives in China, on ARGs in layer feces were estimated by performing metagenomic sequencings and fluorescence quantitative PCR. Fructooligosaccharide significantly reduced sum abundance of ARGs and mobile genetic elements (MGEs) by increasing Lactobacillus clones and reducing Escherichia clones which had relatively higher abundances of ARG subtypes and MGE subtypes in layer feces. However, at least parts of core ARGs and MGEs categories were not reduced by FOS, such as aminoglycosides- and tetracyclines-resistant genes, Tn916, Integrase, and so on. MGEs and microbiome, especially Escherichia genus and Lactobacillus genus, were the key factors affecting ARGs' sum abundance. MGEs had a higher correlation coefficient with ARGs' sum abundance than Escherichia genus and Lactobacillus genus. These findings firstly reveal the defects of antibiotic alternatives in controlling bacterial resistance in livestock and poultry breeding after in-feed antibiotics ban, and more strategies are needed to control pollutions and risks of core ARGs and MGEs in food animals' feces under a special environment.

Stimulating the biofilm formation of Bacillus populations to mitigate soil antibiotic resistome during insect fertilizer application.

Antibiotic resistance in soil introduced by organic fertilizer application pose a globally recognized threat to human health. Insect organic fertilizer may be a promising alternative due to its low antibiotic resistance. However, it is not yet clear how to regulate soil microbes to reduce antibiotic resistance in organic fertilizer agricultural application. In this study, we investigated soil microbes and antibiotic resistome under black soldier fly organic fertilizer (BOF) application in pot and field systems. Our study shows that BOF could stimulate ARB (antibiotic resistant - bacteria) - suppressive Bacillaceae in the soil microbiome and reduce antibiotic resistome. The carbohydrate transport and metabolism pathway of soil Bacillaceae was strengthened, which accelerated the synthesis and transport of polysaccharides to form biofilm to antagonistic soil ARB, and thus reduced the antibiotic resistance. We further tested the ARB - suppressive Bacillus spp. in a microcosm assay, which resulted in a significant decrease in the presence of ARGs and ARB together with higher abundance in key biofilm formation gene (epsA). This knowledge might help to the development of more efficient bio-fertilizers aimed at mitigating soil antibiotic resistance and enhancing soil health, in particular, under the requirements of global "One Health".

Overexpression of Six1 gene suppresses proliferation and enhances expression of fast-type muscle genes in C2C12 myoblasts.

Sine oculis homeobox 1 (Six1) homeodomain transcription factor is implicated in the genesis of muscle fiber type diversity, but its regulatory mechanisms on the formation of muscle fiber type are still poorly understood. To elucidate the biological roles of Six1 gene in muscle fiber formation, we established C2C12 cell line overexpressing Six1 and determined the effects of forced Six1 expression on muscle-specific genes expression, cell proliferation, and cell cycles. Our results indicated that Six1 overexpression could significantly promote the expression of fast-type muscle genes Atp2a1, Srl, and Mylpf. Furthermore, Six1 overexpressing C2C12 cells displayed a relative lower proliferative potential, and cell cycle analysis showed that Six1 exerted its role in cell cycle primarily through the regulation of G1/S and G2/M phases. In conclusion, Six1 plays an essential role in modulation of the fast-twitch muscle fiber phenotype through up-regulating fast-type muscle genes expression, and it could suppress the proliferation of muscle cells.

Lipid droplets: a cellular organelle vital in cancer cells.

Lipid droplets (LDs) are cellular organelles comprising a core of neutral lipids (glycerides, sterols) encased within a single phospholipid membrane, responsible for storing surplus lipids and furnishing cellular energy. LDs engage in lipid synthesis, catabolism, and transport processes by interacting with other organelles (e.g., endoplasmic reticulum, mitochondria), and they play critical roles in regulating cellular stress and immunity. Recent research has uncovered that an elevated number of LDs is a hallmark of cancer cells, attributable to their enhanced lipid uptake and synthesis capacity, with lipids stored as LDs. Depletion of LDs in cancer cells induces apoptosis, prompting the emergence of small molecule antitumor drugs targeting LDs or key factors (e.g., FASN, SCD1) within the lipid synthesis pathway. Advancements in LD isolation and artificial synthesis have demonstrated their potential applicability in antitumor research. LDs extracted from murine adipose tissue and incubated with lipophilic antitumor drugs yield drug-coated LDs, which promote apoptosis in cancer cells. Furthermore, LDs have been employed as biological lenses to augment the resolution of subcellular structures (microfilaments, microtubules), facilitating the observation of intricate structures within thicker cells, including cancer cells. This review delineates the functional and metabolic mechanisms of LDs in cancer cells and encapsulates recent progress in LD-centered antitumor research, offering novel insights for tumor diagnosis and treatment.

Synthetic microbial consortia to enhance the biodegradation of compost odor by biotrickling filter.

Composting generates odorous gases, including ammonia (NH3), hydrogen sulfide (H2S), and volatile organic compounds (VOCs). The Biological Trickling Filter (BTF) is effective for odor treatment, but it may have limitations with hydrophobic VOCs. In this study, a strain of Bacillus subtilis with ammonia-reducing ability, a strain of Bacillus cereus with desulfurization ability and a strain of Schizophyllum commune with the ability to degrade dimethyl disulfide were isolated and screened. The three strains were combined to create synthetic microbial consortia for enhancing odor treatment in the BTF. Compared to the activated sludge control, the BTF with synthetic microbial consortia removed 92.43% ammonia, 92.75% hydrogen sulfide. Furthermore, it demonstrated a significant improvement in the removal rates of p-methyl mercaptan, methyl sulfide, and dimethyl disulfide. High-throughput sequencing was conducted on the fillers of the synthetic microbial consortia-inoculated BTF to analyze the microbial community composition.