Development of engineered Candida tropicalis strain for efficient corncob-based xylitol-ethanol biorefinery.

BACKGROUND: Xylitol has a wide range of applications in the pharmaceuticals, cosmetic, food and beverage industry. Microbial xylitol production reduces the risk of contamination and is considered as environment friendly and sustainable compared to the chemical method. In this study, random mutagenesis and genetic engineering approaches were employed to develop Candida tropicalis strains with reduced xylitol dehydrogenase (XDH) activity to eliminate co-substrate requirement for corn cob-based xylitol-ethanol biorefinery. RESULTS: The results suggest that when pure xylose (10% w/v) was fermented in bioreactor, the Ethyl methane sulfonate (EMS) mutated strain (C. tropicalis K2M) showed 9.2% and XYL2 heterozygous (XYL2/xyl2Δ::FRT) strain (C. tropicalis K21D) showed 16% improvement in xylitol production compared to parental strain (C. tropicalis K2). Furthermore, 1.5-fold improvement (88.62 g/L to 132 g/L) in xylitol production was achieved by C. tropicalis K21D after Response Surface Methodology (RSM) and one factor at a time (OFAT) applied for media component optimization. Finally, corncob hydrolysate was tested for xylitol production in biorefinery mode, which leads to the production of 32.6 g/L xylitol from hemicellulosic fraction, 32.0 g/L ethanol from cellulosic fraction and 13.0 g/L animal feed. CONCLUSIONS: This work, for the first time, illustrates the potential of C. tropicalis K21D as a microbial cell factory for efficient production of xylitol and ethanol via an integrated biorefinery framework by utilising lignocellulosic biomass with minimum waste generation.

Transcriptomic analysis of PADI4 target genes during multi-lineage differentiation of embryonic stem cells.

During mammalian embryo development, pluripotent epiblast cells diversify into the three primary germ layers, which will later give rise to all fetal and adult tissues. These processes involve profound transcriptional and epigenetic changes that require precise coordination. Peptidylarginine deiminase IV (PADI4) is a transcriptional regulator that is strongly associated with inflammation and carcinogenesis but whose physiological roles are less well understood. We previously found that Padi4 expression is associated with pluripotency. Here, we examined the role of PADI4 in maintaining the multi-lineage differentiation potential of mouse embryonic stem (ES) cells. Using bulk and single-cell transcriptomic analyses of embryoid bodies (EBs) derived from Padi4 knock-out (Padi4-KO) mouse ES cells, we find that PADI4 loss impairs mesoderm diversification and differentiation of cardimyocytes and endothelial cells. Additionally, Padi4 deletion leads to concerted downregulation of genes associated with polarized growth, sterol metabolism and the extracellular matrix (ECM). This study indicates a requirement for Padi4 in the specification of the mesodermal lineage and reports the Padi4 associated transcriptome, providing a platform for understanding the physiological functions of Padi4 in development and homeostasis. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.

Surgical Challenges in the Management of Post COVID-19 Midface Mucormycosis (PCoMM): An Institutional Protocol.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic has posed another serious threat, mucormycosis infection, affecting the maxilla and orbitocerebral region. This condition has not spared world population from its merciless claws. This article addresses the challenges faced by the maxillofacial surgeons in setting the protocols from preoperative diagnosis, surgical management to postoperative care, including short-term and long-term rehabilitation. To manage this relentlessly progressing condition, a multispecialty team approach is to be activated in diagnosing, managing, and rehabilitating the patients. PURPOSE: The purpose of this clinical study is to document and analyze the clinical and demographic data, presentation of the lesion, the diagnostic methods followed for early clinical detection, and management of post COVID-19 midface mucormycosis. The article also discusses postoperative medical management and prosthetic rehabilitation. RESULTS: Most of the mucormycosis cases reporting to our center were treated and recovered patients of Severe Acute Respiratory Syndrome Coronavirus 2 infection. Thirty-four (n=34) case were operated for post COVID-19 midface mucormycosis between October 2020 and December 2021. Male to Female ratio is 1:42. The average age of the patients was 57.5 years. Maximum patients were in fifth and sixth decade of life. Maxilla was the involved bone. Treatment was primarily surgical debridement to extended or radical maxillectomy. All patients were treated with Liposomal Amphotericin B and tab posaconazole for 3 to 4 weeks depending upon the age, weight, and physiological state of the patients to attain an optimal cumulative load. Three patients succumbed to illness postoperatively (n=3, 1.02%). Average duration of hospital stay was 47 days. The average review period was 5.1 months.

Congenital Granular Cell Tumour - Case Report and Review of Literature.

Rationale: Congenital orofacial swellings in neonates are mainly limited to vascular malformations and neuroectodermal benign tumours. Congenital granular cell tumour (CGCT) is a rare condition affecting neonates with a prevalence rate of 6 in 1 million. Our report provides a brief review of diagnosis and management. Patient Concern: A 4-day-old female neonate was brought in with the chief complaint of a single, lobulated mass protruding from the right side of the oral cavity. The inability to achieve lip seal and suckling resulting in feeding problems was the primary concern. Diagnosis and Treatment: Surgical excision of the lesion was carried out under general anaesthesia. Resected mass was confirmed to be a CGCT upon histopathological evaluation. Outcome: One-year follow-up showed satisfactory healing with no evidence of recurrence. Take-away Lesson: Ultrasonography and other imaging modalities help in differentiating it from vascular malformations. Simple surgical excision suffices to treat the condition.

The BRCA1 BRCT promotes antisense RNA production and double-stranded RNA formation to suppress ribosomal R-loops.

R-loops, or RNA:DNA hybrids, can induce DNA damage, which requires DNA repair factors including breast cancer type 1 susceptibility protein (BRCA1) to restore genomic integrity. To date, several pathogenic mutations have been found within the tandem BRCA1 carboxyl-terminal (BRCT) domains that mediate BRCA1 interactions with proteins and DNA in response to DNA damage. Here, we describe a nonrepair role of BRCA1 BRCT in suppressing ribosomal R-loops via two mechanisms. Through its RNA binding and annealing activities, BRCA1 BRCT facilitates the formation of double-stranded RNA between ribosomal RNA (rRNA) and antisense-rRNA (as-rRNA), hereby minimizing rRNA hybridization to ribosomal DNA to form R-loops. BRCA1 BRCT also promotes RNA polymerase I-dependent transcription of as-rRNA to enhance double-stranded rRNA (ds-rRNA) formation. In addition, BRCA1 BRCT-mediated as-rRNA production restricts rRNA maturation in unperturbed cells. Hence, impairing as-rRNA transcription and ds-rRNA formation due to BRCA1 BRCT deficiency deregulates rRNA processing and increases ribosomal R-loops and DNA breaks. Our results link ribosomal biogenesis dysfunction to BRCA1-associated genomic instability.

EZH2-H3K27me3 mediated KRT14 upregulation promotes TNBC peritoneal metastasis.

Triple-Negative Breast Cancer (TNBC) has a poor prognosis and adverse clinical outcomes among all breast cancer subtypes as there is no available targeted therapy. Overexpression of Enhancer of zeste homolog 2 (EZH2) has been shown to correlate with TNBC's poor prognosis, but the contribution of EZH2 catalytic (H3K27me3) versus non-catalytic EZH2 (NC-EZH2) function in TNBC progression remains elusive. We reveal that selective hyper-activation of functional EZH2 (H3K27me3) over NC-EZH2 alters TNBC metastatic landscape and fosters its peritoneal metastasis, particularly splenic. Instead of H3K27me3-mediated repression of gene expression; here, it promotes KRT14 transcription by attenuating binding of repressor SP1 to its promoter. Further, KRT14 loss significantly reduces TNBC migration, invasion, and peritoneal metastasis. Consistently, human TNBC metastasis displays positive correlation between H3K27me3 and KRT14 levels. Finally, EZH2 knockdown or H3K27me3 inhibition by EPZ6438 reduces TNBC peritoneal metastasis. Altogether, our preclinical findings suggest a rationale for targeting TNBC with EZH2 inhibitors.

Melanotic Neuroectodermal Tumour of Infancy - A Case Report and Review of Literature.

Rationale: Melanotic neuroectodermal tumour of infancy (MNTI) is universally described as a rare, benign, pigmented lesion which most frequently involves the maxilla. Its origin is well established to be in the neural crest cells. Due to the high recurrence rate and aggressive behaviour mimicking malignancy, it poses a great challenge in their diagnosis, treatment planning, and prognosis. Patient Concern: Two-year-old female with no known comorbidities was brought in with the chief complaint of a growing swelling in the upper lip region. Diagnosis and Treatment: She was taken up for resection of the tumour under general anaesthesia. The specimen was subjected to histological and immunological examination confirming the diagnosis of MNTI. Outcome: The postoperative period was uneventful. After regular follow-up, the patient showed satisfactory healing with no signs of recurrence. Take-Away Lessons: Based on our experience, we feel that the diagnosis of MNTI is mainly clinical. Early conservative surgical excision and regular follow-up provide an excellent result with good prognosis.

Improved xylitol production by the novel inhibitor-tolerant yeast Candida tropicalis K2.

HIGHLIGHTSCandia tropicalis K2 isolate was screened from natural sites of biomass degradation and characterized for xylitol production.Non-detoxified Albizia pod and corncob hydrolysates were explored for xylitol production using selected C. tropicalis K2 isolate.A maximum of 0.90 g/g yield and 1.07 g/L.h xylitol productivity was achieved with pure xylose.A >10% increase in xylitol yield was achieved using glycerol as a co-substrate.

Time versus tissue: Timely identification of Scedosporium Rhinosinusitis in a post-COVID-19 case by MALDI-TOF MS leading to successful management.

COVID-19 (Coronavirus Disease 2019), illness with associated comorbidities and corticosteroid therapy makes the host immunocompromised and prone to opportunistic microbial infections. As the world continues to struggle with the pandemic of COVID-19, an increase in cases of opportunistic fungal infections have been reported from all over the world during the second wave of COVID-19 like aspergillosis, mucormycosis, and candidiasis. Scedosporium apiospermum is an emerging pathogen that is usually associated with mycetoma, pulmonary infection, and central nervous infections. It has been rarely associated with fungal rhinosinusitis (FRS). In this study, a rare case of FRS caused by S.apiospermum in an immunocompromised post-Covid-19 diabetic woman is reported.

Characterization of Arsenic-Resistant Klebsiella pneumoniae RnASA11 from Contaminated Soil and Water Samples and Its Bioremediation Potential.

Rapid industrialization and intensive agriculture activities have led to a rise in heavy metal contamination all over the world. Chhattisgarh (India) being an industrial state, the soil and water are thickly contaminated with heavy metals, especially from arsenic (As). In the present study, we isolated 108 arsenic-resistant bacteria (both from soil and water) from different arsenic-contaminated industrial and mining sites of Chhattisgarh to explore the bacterial gene pool. Further, we screened 24 potential isolates out of 108 for their ability to tolerate a high level of arsenic. The sequencing of the 16S rRNA gene of bacterial isolates revealed that all these samples belong to different diverse genera including Bacillus, Enterobacter, Klebsiella, Pantoea, Acinetobacter, Cronobacter, Pseudomonas and Agrobacterium. The metal tolerance ability was determined by amplification of arsB (arsenite efflux gene) and arsC (arsenate reductase gene) from chromosomal DNA of isolated RnASA11, which was identified as Klebsiella pneumoniae through in silico analysis. The bacterial strains RpSWA2 and RnASA11 were found to tolerate 600 mM As (V) and 30 mM As (III) but the growth of strain RpSWA2 was slower than RnASA11. Furthermore, atomic absorption spectroscopy (AAS) of the sample obtained from bioremediation assay revealed that Klebsiella pneumoniae RnASA11 was able to reduce the arsenic concentration significantly in the presence of arsenate (44%) and arsenite (38.8%) as compared to control.

Virus-Encoded Complement Regulators: Current Status.

Viruses require a host for replication and survival and hence are subjected to host immunological pressures. The complement system, a crucial first response of the host immune system, is effective in targeting viruses and virus-infected cells, and boosting the antiviral innate and acquired immune responses. Thus, the system imposes a strong selection pressure on viruses. Consequently, viruses have evolved multiple countermeasures against host complement. A major mechanism employed by viruses to subvert the complement system is encoding proteins that target complement. Since viruses have limited genome size, most of these proteins are multifunctional in nature. In this review, we provide up to date information on the structure and complement regulatory functions of various viral proteins.

Salinomycin inhibits epigenetic modulator EZH2 to enhance death receptors in colon cancer stem cells.

Drug resistance is one of the trademark features of Cancer Stem Cells (CSCs). We and others have recently shown that paucity of functional death receptors (DR4/5) on the cell surface of tumour cells is one of the major reasons for drug resistance, but their involvement in the context of in CSCs is poorly understood. By harnessing CSC specific cytotoxic function of salinomycin, we discovered a critical role of epigenetic modulator EZH2 in regulating the expression of DRs in colon CSCs. Our unbiased proteome profiler array approach followed by ChIP analysis of salinomycin treated cells indicated that the expression of DRs, especially DR4 is epigenetically repressed in colon CSCs. Concurrently, EZH2 knockdown demonstrated increased expression of DR4/DR5, significant reduction of CSC phenotypes such as spheroid formation in-vitro and tumorigenic potential in-vivo in colon cancer. TCGA data analysis of human colon cancer clinical samples shows strong inverse correlation between EZH2 and DR4. Taken together, this study provides an insight about epigenetic regulation of DR4 in colon CSCs and advocates that drug-resistant colon cancer can be therapeutically targeted by combining TRAIL and small molecule EZH2 inhibitors.

Arsenic efflux in Enterobacter cloacae RSN3 isolated from arsenic-rich soil.

In the present study, bacterial isolates were screened for arsenic resistance efficiency. Environmental isolates were isolated from arsenic-rich soil samples (i.e., from Rajnandgaon district of Chhattisgarh state, India). Amplification and sequencing of 16S rRNA gene revealed that the isolates were of Bacillus firmus RSN1, Brevibacterium senegalense RSN2, Enterobacter cloacae RSN3, Stenotrophomonas pavanii RSN6, Achromobacter mucicolens RSN7, and Ochrobactrum intermedium RSN10. Arsenite efflux gene (arsB) was successfully amplified in E. cloacae RSN3. Atomic absorption spectroscopy (AAS) analysis showed an absorption of 32.22% arsenic by the RSN3 strain. Furthermore, results of scanning electron microscopy (SEM) for morphological variations revealed an initial increase in the cell size at 1 mM sodium arsenate; however, it was decreased at 10 mM concentration in comparison to control. This change of the cell size in different metal concentrations was due to the uptake and expulsion of the metal from the cell, which also confirmed the arsenite efflux system.

Tissue-Specific Carcinogens as Soil to Seed BRCA1/2-Mutant Hereditary Cancers.

Despite their ubiquitous expression, the inheritance of monoallelic germline mutations in breast cancer susceptibility gene type 1 or 2 (BRCA1/2) poses tissue-specific variations in cancer risks and primarily associate with familial breast and ovarian cancers. The molecular basis of this tissue-specific tumor incidence remains unknown and intriguing to cancer researchers. A plethora of recent reports support the idea that several nongenetic factors present in the tissue microenvironment could induce tumors in the mutant BRCA1/2 background. This Opinion article summarizes the recent advances on tissue-specific carcinogens and their complex crosstalk with the compromised DNA repair machinery of BRCA1/2-mutant cells. Finally, we present our perspective on the therapeutic and chemopreventive interpretations of these developments.

Selective targeting of TET catalytic domain promotes somatic cell reprogramming.

Ten-eleven translocation (TET) family enzymes (TET1, TET2, and TET3) oxidize 5-methylcytosine (5mC) and generate 5-hydroxymethylcytosine (5hmC) marks on the genome. Each TET protein also interacts with specific binding partners and partly plays their role independent of catalytic activity. Although the basic role of TET enzymes is well established now, the molecular mechanism and specific contribution of their catalytic and noncatalytic domains remain elusive. Here, by combining in silico and biochemical screening strategy, we have identified a small molecule compound, C35, as a first-in-class TET inhibitor that specifically blocks their catalytic activities. Using this inhibitor, we explored the enzymatic function of TET proteins during somatic cell reprogramming. Interestingly, we found that C35-mediated TET inactivation increased the efficiency of somatic cell programming without affecting TET complexes. Using high-throughput mRNA sequencing, we found that by targeting 5hmC repressive marks in the promoter regions, C35-mediated TET inhibition activates the transcription of the BMP-SMAD-ID signaling pathway, which may be responsible for promoting somatic cell reprogramming. These results suggest that C35 is an important tool for inducing somatic cell reprogramming, as well as for dissecting the other biological functions of TET enzymatic activities without affecting their other nonenzymatic roles.

Poly(ADP-ribosyl)ation mediates early phase histone eviction at DNA lesions.

Nucleosomal histones are barriers to the DNA repair process particularly at DNA double-strand breaks (DSBs). However, the molecular mechanism by which these histone barriers are removed from the sites of DNA damage remains elusive. Here, we have generated a single specific inducible DSB in the cells and systematically examined the histone removal process at the DNA lesion. We found that histone removal occurred immediately following DNA damage and could extend up to a range of few kilobases from the lesion. To examine the molecular mechanism underlying DNA damage-induced histone removal, we screened histone modifications and found that histone ADP-ribosylation was associated with histone removal at DNA lesions. PARP inhibitor treatment suppressed the immediate histone eviction at DNA lesions. Moreover, we examined histone chaperones and found that the FACT complex recognized ADP-ribosylated histones and mediated the removal of histones in response to DNA damage. Taken together, our results reveal a pathway that regulates early histone barrier removal at DNA lesions. It may also explain the mechanism by which PARP inhibitor regulates early DNA damage repair.

Identification of Rpl29 as a major substrate of the lysine methyltransferase Set7/9.

Set7/9 (also known as Set7, Set9, Setd7, and Kmt7) is a lysine methyltransferase that catalyzes the methylation of multiple substrates, including histone H3 and non-histone proteins. Although not essential for normal development and physiology, Set7/9-mediated methylation events play important roles in regulating cellular pathways involved in various human diseases, making Set7/9 a promising therapeutic target. Multiple Set7/9 inhibitors have been developed, which exhibit varying degrees of potency and selectivity in vitro However, validation of these compounds in vivo has been hampered by the lack of a reliable cellular biomarker for Set7/9 activity. Here, we report the identification of Rpl29, a ribosomal protein abundantly expressed in all cell types, as a major substrate of Set7/9. We show that Rpl29 lysine 5 (Rpl29K5) is methylated exclusively by Set7/9 and can be demethylated by Lsd1 (also known as Kdm1a). Rpl29 is not a core component of the ribosome translational machinery and plays a regulatory role in translation efficiency. Our results indicate that Rpl29 methylation has no effect on global protein synthesis but affects Rpl29 subcellular localization. Using an Rpl29 methylation-specific antibody, we demonstrate that Rpl29K5 methylation is present ubiquitously and validate that (R)-PFI-2, a Set7/9 inhibitor, efficiently reduces Rpl29K5 methylation in cell lines. Thus, Rpl29 methylation can serve as a specific cellular biomarker for measuring Set7/9 activity.

Self-Assembled Spheres, Flowers, and Fibers from the Same Backbone and Similar Side Chains.

Rylene imides (RIs) self-assemble into various nanostructures. Often, the synthesis of unsymmetrical RIs (URIs) is required to achieve nanostructures. However, the synthesis of URIs is nontrivial. Thus, a structurally similar alternative is desirable. iso-Indigo (i-indigo) has a π core and lactam rings that are structurally similar to the RIs. Unsymmetrical iso-indigo (i-indigo) can be easily synthesized by condensing oxindole and isatin. We have synthesized a series of unsymmetrical i-indigo molecules. In these molecules, the π-π interaction, hydrogen bonding, and van der Waals interactions are in operation. Because of these, the molecules self-assemble into spheres, fibers, and dahlia flower morphologies. If the hydrogen bonding interaction is disrupted, then all of them form fibers. Control experiments indicate that the complete absence of hydrogen bonding is deleterious to self-assembly. We also show that the lower analogs of i-indigo are not sufficient to form self-assembled nanostructures.

A WRKY transcription factor from Withania somnifera regulates triterpenoid withanolide accumulation and biotic stress tolerance through modulation of phytosterol and defense pathways.

Withania somnifera produces pharmacologically important triterpenoid withanolides that are derived via phytosterol pathway; however, their biosynthesis and regulation remain to be elucidated. A jasmonate- and salicin-inducible WRKY transcription factor from W. somnifera (WsWRKY1) exhibiting correlation with withaferin A accumulation was functionally characterized employing virus-induced gene silencing and overexpression studies combined with transcript and metabolite analyses, and chromatin immunoprecipitation assay. WsWRKY1 silencing resulted in stunted plant growth, reduced transcripts of phytosterol pathway genes with corresponding reduction in phytosterols and withanolides in W. somnifera. Its overexpression elevated the biosynthesis of triterpenoids in W. somnifera (phytosterols and withanolides), as well as tobacco and tomato (phytosterols). Moreover, WsWRKY1 binds to W-box sequences in promoters of W. somnifera genes encoding squalene synthase and squalene epoxidase, indicating its direct regulation of triterpenoid pathway. Furthermore, while WsWRKY1 silencing in W. somnifera compromised the tolerance to bacterial growth, fungal infection, and insect feeding, its overexpression in tobacco led to improved biotic stress tolerance. Together these findings demonstrate that WsWRKY1 has a positive regulatory role on phytosterol and withanolides biosynthesis, and defense against biotic stress, highlighting its importance as a metabolic engineering tool for simultaneous improvement of triterpenoid biosynthesis and plant defense.

Nitrogen treatment enhances sterols and withaferin A through transcriptional activation of jasmonate pathway, WRKY transcription factors, and biosynthesis genes in Withania somnifera (L.) Dunal.

The medicinal plant Withania somnifera is researched extensively to increase the quantity of withanolides and specifically withaferin A, which finds implications in many pharmacological activities. Due to insufficient knowledge on biosynthesis and unacceptability of transgenic approach, it is preferred to follow alternative physiological methods to increase the yield of withanolides. Prior use of elicitors like salicylic acid, methyl jasmonate, fungal extracts, and even mechanical wounding have shown to increase the withanolide biosynthesis with limited success; however, the commercial viability and logistics of application are debatable. In this investigation, we tested the simple nitrogeneous fertilizers pertaining to the enhancement of withaferin A biosynthesis. Application of ammonium sulfate improved the sterol contents required for the withanolide biosynthesis and correlated to higher expression of pathway genes like FPPS, SMT1, SMT2, SMO1, SMO2, and ODM. Increased expression of a gene homologous to allene oxide cyclase, crucial in jasmonic acid biosynthetic pathway, suggested the involvement of jasmonate signaling. High levels of WRKY gene transcripts indicated transcriptional regulation of the pathway genes. Increase in transcript level could be correlated with a corresponding increase in the protein levels for WsSMT1 and WsWRKY1. The withaferin A increase was also demonstrated in the potted plants growing in the glasshouse and in the open field. These results implicated simple physiological management of nitrogen fertilizer signal to improve the yield of secondary metabolite through probable involvement of jasmonate signal and WRKY transcription factor for the first time, in W. somnifera besides improving the foliage.