Two dd-Carboxypeptidases from Mycobacterium smegmatis Affect Cell Surface Properties through Regulation of Peptidoglycan Cross-Linking and Glycopeptidolipids.

During the peptidoglycan (PG) maturation of mycobacteria, the glycan strands are interlinked by both 3-3 (between two meso-diaminopimelic acids [meso-DAPs]) and 4-3 cross-links (between d-Ala and meso-DAP), though there is a predominance (60 to 80%) of 3-3 cross-links. The dd-carboxypeptidases (dd-CPases) act on pentapeptides to generate tetrapeptides that are used by ld-transpeptidases as substrates to form 3-3 cross-links. Therefore, dd-CPases play a crucial role in mycobacterial PG cross-link formation. However, the physiology of dd-CPases in mycobacteria is relatively unexplored. In this study, we deleted two dd-CPase genes, msmeg_2433 and msmeg_2432, both individually and in combination, from Mycobacterium smegmatis mc2155. Though the single dd-CPase gene deletions had no significant impact on the mycobacterial physiology, many interesting functional alterations were observed in the double-deletion mutant, viz, a predominance in PG cross-link formation was shifted from 3-3 cross-links to 4-3, cell surface glycopeptidolipid (GPL) expression was reduced, and susceptibility to ß-lactams and antitubercular agents was enhanced. Moreover, the survival rate of the double mutant within murine macrophages was higher than that of the parent. Interestingly, the complementation with any one of the dd-CPase genes could restore the wild-type phenotype. In a nutshell, we infer that the altered ratio of 4-3 to 3-3 PG cross-links might have influenced the expression of surface GPLs, colony morphology, biofilm formation, drug susceptibility, and subsistence of the cells within macrophages.IMPORTANCE The glycan strands in mycobacterial peptidoglycan (PG) are interlinked by both 3-3 and 4-3 cross-links. The dd-CPases generate tetrapeptides by acting on the pentapeptides, and ld-transpeptidases use tetrapeptides as substrates to form 3-3 cross-links. In this study, we showed that simultaneous deletions of two dd-CPases alter the nature of PG cross-linking from 3-3 cross-links to 4-3 cross-links. The deletions subsequently decrease the expression of glycopeptidolipids (significant surface lipid present in many nontuberculous mycobacteria, including Mycobacterium smegmatis) and affect other physiological parameters, like cell morphology, growth rate, biofilm formation, antibiotic susceptibility, and survival within murine macrophages. Thus, unraveling the physiology of dd-CPases might help us design antimycobacterial therapeutics in the future.

Uncoupling lifespan and healthspan in Caenorhabditis elegans longevity mutants.

Aging research has been very successful at identifying signaling pathways and evolutionarily conserved genes that extend lifespan with the assumption that an increase in lifespan will also increase healthspan. However, it is largely unknown whether we are extending the healthy time of life or simply prolonging a period of frailty with increased incidence of age-associated diseases. Here we use Caenorhabditis elegans, one of the premiere systems for lifespan studies, to determine whether lifespan and healthspan are intrinsically correlated. We conducted multiple cellular and organismal assays on wild type as well as four long-lived mutants (insulin/insulin-like growth factor-1, dietary restriction, protein translation, mitochondrial signaling) in a longitudinal manner to determine the health of the animals as they age. We find that some long-lived mutants performed better than wild type when measured chronologically (number of days). However, all long-lived mutants increased the proportion of time spent in a frail state. Together, these data suggest that lifespan can no longer be the sole parameter of interest and reveal the importance of evaluating multiple healthspan parameters for future studies on antiaging interventions.

Bioremediation of steel plant wastewater and enhanced electricity generation in microbial desalination cell.

Microbial desalination cell (MDC) is a propitious technology towards water desalination by utilizing wastewater as an energy source. In this study, a multi-chambered MDC was used to bioremediate steel plant wastewater using the same wastewater as a fuel for anodic bacteria. A pure culture of Pseudomonas putida MTCC 1194 was isolated and inoculated to remove toxic phenol. Three different inoculum conditions, namely P. putida (INC-A), a mixture of P. putida and activated sludge (INC-B), and activated sludge alone (INC-C) were employed in an anodic chamber to mainly compare the electricity generation and phenol degradation in MDCs. The study revealed the maximum phenol removal of 82 ± 2.4%, total dissolved solids (TDS) removal of 68 ± 1.5%, and power generation of 10.2 mW/m2 using INC-B. The synergistic interactions between microorganisms, can enhance the toxic phenol degradation and also electricity generation in MDC for onsite wastewater application.

A single amino acid substitution in the Ω-like loop of E. coli PBP5 disrupts its ability to maintain cell shape and intrinsic beta-lactam resistance.

Penicillin-binding protein 5 (PBP5), a dd-carboxypeptidase, maintains cell shape and intrinsic beta-lactam resistance in E. coli. A strain lacking PBP5 loses intrinsic beta-lactam resistance and simultaneous lack of two other PBPs results in aberrantly shaped cells. PBP5 expression in trans complements the shape and restores the lost beta-lactam resistance. PBP5 has an 'Ω-loop'-like region similar to that in class A beta-lactamases. It was previously predicted that Leu182 present in the 'Ω-like' loop of PBP5 corresponds to Glu166 in PER-1 beta-lactamase. Here, we studied the physiological and biochemical effects of the Leu182Glu mutation in PBP5. Upon overexpression in septuple PBP mutants, ~75 % of cells were abnormally shaped and intrinsic beta-lactam resistance maintenance was partially lost. Biochemically, the purified soluble mutated PBP5 (smPBP5) retained low acylation ability for penicillin. The turnover number of smPBP5 for artificial and peptidoglycan mimetic substrates was ~10-fold less than that of the wild-type. Superimposition of the active-site residues of smPBP5 on PBP5 revealed that perturbation in the orientating key residues may explain the low turnover numbers. Therefore, we establish the involvement of Leu182 in maintaining the physiological and biochemical behaviour of E. coli PBP5.

A putative low-molecular-mass penicillin-binding protein (PBP) of Mycobacterium smegmatis exhibits prominent physiological characteristics of DD-carboxypeptidase and beta-lactamase.

DD-carboxypeptidases (DD-CPases) are low-molecular-mass (LMM) penicillin-binding proteins (PBPs) that are mainly involved in peptidoglycan remodelling, but little is known about the dd-CPases of mycobacteria. In this study, a putative DD-CPase of Mycobacterium smegmatis, MSMEG_2433 is characterized. The gene for the membrane-bound form of MSMEG_2433 was cloned and expressed in Escherichia coli in its active form, as revealed by its ability to bind to the Bocillin-FL (fluorescent penicillin). Interestingly, in vivo expression of MSMEG_2433 could restore the cell shape oddities of the septuple PBP mutant of E. coli, which was a prominent physiological characteristic of DD-CPases. Moreover, expression of MSMEG_2433 in trans elevated beta-lactam resistance in PBP deletion mutants (ΔdacAdacC) of E. coli, strengthening its physiology as a dd-CPase. To confirm the biochemical reason behind such physiological behaviours, a soluble form of MSMEG_2433 (sMSMEG_2433) was created, expressed and purified. In agreement with the observed physiological phenomena, sMSMEG_2433 exhibited DD-CPase activity against artificial and peptidoglycan-mimetic DD-CPase substrates. To our surprise, enzymic analyses of MSMEG_2433 revealed efficient deacylation for beta-lactam substrates at physiological pH, which is a unique characteristic of beta-lactamases. In addition to the MSMEG_2433 active site that favours dd-CPase activity, in silico analyses also predicted the presence of an omega-loop-like region in MSMEG_2433, which is an important determinant of its beta-lactamase activity. Based on the in vitro, in vivo and in silico studies, we conclude that MSMEG_2433 is a dual enzyme, possessing both DD-CPase and beta-lactamase activities.

PDP-1 links the TGF-ß and IIS pathways to regulate longevity, development, and metabolism.

The insulin/IGF-1 signaling (IIS) pathway is a conserved regulator of longevity, development, and metabolism. In Caenorhabditis elegans IIS involves activation of DAF-2 (insulin/IGF-1 receptor tyrosine kinase), AGE-1 (PI 3-kinase), and additional downstream serine/threonine kinases that ultimately phosphorylate and negatively regulate the single FOXO transcription factor homolog DAF-16. Phosphatases help to maintain cellular signaling homeostasis by counterbalancing kinase activity. However, few phosphatases have been identified that negatively regulate the IIS pathway. Here we identify and characterize pdp-1 as a novel negative modulator of the IIS pathway. We show that PDP-1 regulates multiple outputs of IIS such as longevity, fat storage, and dauer diapause. In addition, PDP-1 promotes DAF-16 nuclear localization and transcriptional activity. Interestingly, genetic epistasis analyses place PDP-1 in the DAF-7/TGF-ß signaling pathway, at the level of the R-SMAD proteins DAF-14 and DAF-8. Further investigation into how a component of TGF-ß signaling affects multiple outputs of IIS/DAF-16, revealed extensive crosstalk between these two well-conserved signaling pathways. We find that PDP-1 modulates the expression of several insulin genes that are likely to feed into the IIS pathway to regulate DAF-16 activity. Importantly, dysregulation of IIS and TGF-ß signaling has been implicated in diseases such as Type 2 Diabetes, obesity, and cancer. Our results may provide a new perspective in understanding of the regulation of these pathways under normal conditions and in the context of disease.

Differential vertebral body growth is maintained after vertebral body tethering surgery for idiopathic scoliosis: 4-year follow-up on 888 peri-apical vertebrae and 592 intervertebral discs.

PURPOSE: To radiographically evaluate if vertebral body tethering (VBT) can maintain differential peri-apical vertebral growth at medium-term follow-up of 4 years. METHODS: A prospective, international, multicenter database was queried to identify idiopathic scoliosis patients treated with thoracic VBT. Concave vs. convex vertebral body height, vertebral wedging, and disc wedging of the 3 peri-apical vertebrae were measured by two independent observers at 5 timepoints (pre-operative to 4-year follow-up). RESULTS: 65 skeletally immature patients (60 female, mean 12.8 years old, 21 with open triradiate cartilages) met inclusion criteria. Mean pre-operative maximum scoliosis of 50 ± 8° decreased significantly post-operatively to 27 ± 9° (p < 0.001), which remained stable at 4-year follow-up 30 ± 17° (p = 0.38 vs. post-operative). Mean instrumented scoliosis was 21 ± 14° at 4-year follow-up, which was significantly different than 4-year maximum scoliosis (p < 0.001). Mean pre-operative kyphosis of 30 ± 12° did not significantly change post-operatively (p = 1.0) and remained stable at 4-year follow-up (35 ± 18°; p = 0.05). Mean individual convex vertebral height increased from 17.7 ± 1.9 mm to 19.8 ± 1.5 mm (p < 0.001), while mean individual concave height increased from 14.8 ± 1.9 mm to 17.6 ± 1.6 mm (p < 0.001). Summing the peri-apical heights, the difference in height from pre-operative to 4-year follow-up was greater on the concave (8.3 ± 4.7 mm) than on the convex side (6.2 ± 4.7 mm) (p < 0.001). Mean individual vertebral wedging decreased from 6 ± 2° at pre-operative to 4 ± 2° at 4-year follow-up (p < 0.001). Mean total vertebral and disc wedging started at 29 ± 7° pre-operatively, decreased to 16 ± 6° at post-operative (p < 0.001), then further decreased to 14 ± 8° at 4-year follow-up (p < 0.001). Patients with open triradiate cartilages at the time of surgery had a larger height change over the 4 years compared to those with closed triradiate cartilages (p < 0.001). CONCLUSION: Patients with idiopathic scoliosis treated with VBT demonstrated differential vertebral growth which was maintained at minimum 4-year follow-up. This effect was more pronounced in patients whose triradiate cartilages were open at the time of surgery. LEVEL OF EVIDENCE: III.

B12 Deficiency is the Commonest Cause of Anaemia During Pregnancy in Northern India: Study from a Tertiary Care Institute.

Iron deficiency anemia is considered the leading cause of anemia during pregnancy; however, there is a lack of comprehensive studies on the etiological factors of anemia in pregnant women. The objective of this study was to systematically investigate the causes of anemia in pregnancy. Five hundred women with hemoglobin levels < 11 g/dl between 6 and 40 weeks of pregnancy underwent a complete hemogram, iron studies, serum folate, serum B12, serum copper, and serum zinc level assessments using standard methods. The median age of the patients was 26 years (range 24-29 years). The majority of patients were in the third trimester (449/500, 89.8%). Among the patients, 325 (65%) had vitamin B12 deficiency, with 159 (31.8%) having isolated B12 deficiency and 142 (28.4%) having combined B12 and iron deficiency. Isolated iron deficiency anemia was present in 74 patients (14.8%). Additionally, 28 patients (5.6%) had beta-thalassemia minor, and anemia of chronic disease was found in 17.2% (86) of the patients. Vitamin B12 deficiency was the most common cause of anemia, followed by combined B12 and iron deficiency. Further studies in diverse populations are warranted as they have broader implications for nutrient supplementation during pregnancy. Supplementary Information: The online version contains supplementary material available at 10.1007/s12288-023-01682-x.

Aspect-Based Sentiment Analysis Using Attribute Extraction of Hospital Reviews.

The Covid pandemic has become a serious public health challenge for people across India and other nations. Nowadays, people rely on the online reviews being shared on different review sites to gather information about hospitals like the availability of beds, availability of ventilators, etc. However, since these reviews are large in number and are unstructured, patients struggle to get accurate and reliable information about the hospitals, due to which they end up taking admission into a hospital which might not be appropriate for the specific treatment they require. This paper employs the use of sentiment analysis to understand various online reviews of hospitals and provide valuable information to the patients. Approximately 30,000 + reviews were collected from more than 500 hospitals. The broad objective of the study is to give the patients a comprehensive and descriptive rating of the hospitals based on the online reviews given by different patients. In addition to providing a comprehensive summary, the study has conducted aspect-based analysis where it compares the hospitals based on four different aspects of the hospital viz. "Doctors' services", "Staff's services", "Hospital facilities", and "Affordability". The database containing aspect-based ratings of the hospitals will be of great value to the patients by allowing them to compare and select the best hospital based on the optimum fit of the aspects of their preference.

Loss of PBRM1 Alters Promoter Histone Modifications and Activates ALDH1A1 to Drive Renal Cell Carcinoma.

Subunits of SWI/SNF chromatin remodeling complexes are frequently mutated in human malignancies. The PBAF complex is composed of multiple subunits, including the tumor-suppressor protein PBRM1 (BAF180), as well as ARID2 (BAF200), that are unique to this SWI/SNF complex. PBRM1 is mutated in various cancers, with a high mutation frequency in clear cell renal cell carcinoma (ccRCC). Here, we integrate RNA-seq, histone modification ChIP-seq, and ATAC-seq data to show that loss of PBRM1 results in de novo gains in H3K4me3 peaks throughout the epigenome, including activation of a retinoic acid biosynthesis and signaling gene signature. We show that one such target gene, ALDH1A1, which regulates a key step in retinoic acid biosynthesis, is consistently upregulated with PBRM1 loss in ccRCC cell lines and primary tumors, as well as non-malignant cells. We further find that ALDH1A1 increases the tumorigenic potential of ccRCC cells. Using biochemical methods, we show that ARID2 remains bound to other PBAF subunits after loss of PBRM1 and is essential for increased ALDH1A1 after loss of PBRM1, whereas other core SWI/SNF components are dispensable, including the ATPase subunit BRG1. In total, this study uses global epigenomic approaches to uncover novel mechanisms of PBRM1 tumor suppression in ccRCC. IMPLICATIONS: This study implicates the SWI/SNF subunit and tumor-suppressor PBRM1 in the regulation of promoter histone modifications and retinoic acid biosynthesis and signaling pathways in ccRCC and functionally validates one such target gene, the aldehyde dehydrogenase ALDH1A1.

A local tumor microenvironment acquired super-enhancer induces an oncogenic driver in colorectal carcinoma.

Tumors exhibit enhancer reprogramming compared to normal tissue. The etiology is largely attributed to cell-intrinsic genomic alterations. Here, using freshly resected primary CRC tumors and patient-matched adjacent normal colon, we find divergent epigenetic landscapes between CRC tumors and cell lines. Intriguingly, this phenomenon extends to highly recurrent aberrant super-enhancers gained in CRC over normal. We find one such super-enhancer activated in epithelial cancer cells due to surrounding inflammation in the tumor microenvironment. We restore this super-enhancer and its expressed gene, PDZK1IP1, following treatment with cytokines or xenotransplantation into nude mice, thus demonstrating cell-extrinsic etiology. We demonstrate mechanistically that PDZK1IP1 enhances the reductive capacity CRC cancer cells via the pentose phosphate pathway. We show this activation enables efficient growth under oxidative conditions, challenging the previous notion that PDZK1IP1 acts as a tumor suppressor in CRC. Collectively, these observations highlight the significance of epigenomic profiling on primary specimens.

Functional and Radiological Outcomes Following Calcaneo-Cuboid-Cuneiform Osteotomy for the Treatment of Planovalgus Feet: A Short-Term Analysis.

INTRODUCTION: Flexible flatfoot refers to the loss of the medial longitudinal arch of the foot on weight bearing and is associated with excessive heel eversion or forefoot abduction. Unless symptomatic, flexible flatfeet are best managed non-operatively. The calcaneo-cuboid-cuneiform osteotomy is a procedure that restores the anatomical shape of the foot without arthrodesis of the joints. Our study aims to evaluate the functional and radiological outcomes of patients treated with calcaneo-cuboid-cuneiform osteotomy in patients with planovalgus feet. METHODOLOGY: A retrospective review of records and radiographs of patients with symptomatic flexible planovalgus feet, who were operated with the calcaneo-cuboid-cuneiform osteotomy by a single senior surgeon in a time period between April 2016 and July 2017 was done. The clinical and radiological outcomes were evaluated in 12 feet in 8 children. RESULTS: A total of 12 feet in 8 children were operated (6 males and 2 females). Average age of patients was 11 ± 1.27 years; average follow up was 14.7 months ± 2.7 months. Two patients had planovalgus feet secondary to spastic diplegia and 6 had idiopathic planovalgus feet. There was a statistically significant improvement in the pain score as well as the radiographic parameters in all the operated patients. CONCLUSION: The calcaneo-cuboid-cuneiform osteotomy has potential to give good results for symptomatic planovalgus feet with minimal complications.

AKT Degradation Selectively Inhibits the Growth of PI3K/PTEN Pathway-Mutant Cancers with Wild-Type KRAS and BRAF by Destabilizing Aurora Kinase B.

Using a panel of cancer cell lines, we characterized a novel degrader of AKT, MS21. In mutant PI3K-PTEN pathway cell lines, AKT degradation was superior to AKT kinase inhibition for reducing cell growth and sustaining lower signaling over many days. AKT degradation, but not kinase inhibition, profoundly lowered Aurora kinase B (AURKB) protein, which is known to be essential for cell division, and induced G2-M arrest and hyperploidy. PI3K activated AKT phosphorylation of AURKB on threonine 73, which protected it from proteasome degradation. A mutant of AURKB (T73E) that mimics phosphorylation and blocks degradation rescued cells from growth inhibition. Degrader-resistant lines were associated with low AKT phosphorylation, wild-type PI3K/PTEN status, and mutation of KRAS/BRAF. Pan-cancer analysis identified that 19% of cases have PI3K-PTEN pathway mutation without RAS pathway mutation, suggesting that these patients with cancer could benefit from AKT degrader therapy that leads to loss of AURKB. SIGNIFICANCE: MS21 depletes cells of phosphorylated AKT (pAKT) and a newly identified AKT substrate, AURKB, to inhibit tumor growth in mice. MS21 is superior to prior agents that target PI3K and AKT due to its ability to selectively target active, pAKT and sustain repression of signaling to deplete AURKB. This article is highlighted in the In This Issue feature, p. 2945.

Widely Divergent Congenital Inferior Tibiofibular Diastasis with Separate Soft-Tissue Cover and Persistent Sciatic Artery: A Previously Unreported Combination: A Case Report.

CASE: We report a case of a child with widely divergent congenital inferior tibiofibular diastasis with persistent sciatic artery (PSA). The dysplastic tibia and fibula were widely divergent, and the fibula was displaced proximally and medially with the foot alongside the thigh between the 2 legs, with PSA diagnosed on computed tomography angiogram. The child was treated with fibula-foot complex excision and below-knee prosthesis and was ambulating independently at 1-year follow-up. CONCLUSIONS: The combination of a major structural anomaly (tibiofibular diastasis with a separate soft-tissue cover) and an unusual vascular malformation (PSA) has not been reported previously and made surgical reconstruction challenging.

Short Term Results of a New Comprehensive Protocol for the Management of Congenital Pseudarthrosis of the Tibia.

BACKGROUND: Treatment of Congenital Psuedarthrosis of Tibia (CPT) often poses significant challenges due to difficulty in achieving union and subsequent complications like refractures, implant failures, etc. Our new comprehensive protocol is aimed at achieving crossunion between the tibia and fibula. AIMS AND OBJECTIVES: The aim of the present study is to evaluate the short-term results of our new protocol and to compare the results with our previously used techniques. MATERIALS AND METHODS: 10 patients with mean age 2.35 years (1 to 6.5 years) who were treated by our new comprehensive protocol were included in Group A, and 11 patients with mean age 2 years (1 to 5.5 years) who primarily underwent intramedullary rodding with bone graft were included in Group B. The new comprehensive protocol consisted of pre-operative Zolendronate infusion, surgery consisting of intramedullary fixation of tibia supplemented with Ilizarov ring fixator and bone grafting aimed at achieving tibia-fibula cross-union. Retrospective evaluation of serial radiographs was performed and outcomes with respect to union and subsequent complications were analysed. RESULTS: 10/10 (100%) patients in Group A united, whereas union was achieved in only 8/11 (72%) patients in Group B. The index surgery was successful in achieving union in all 10 patients in Group A, whereas in Group B 2.25 (1 to 4) surgeries were needed to achieve union. The time to union was significantly shorter in Group A (4.68 months) as compared to Group B (30.88 months). The cross sectional area of union was significantly greater in Group A (3.82 cm2) as compared to Group B (1.18 cm2). One patient in Group A needed a subsequent corrective osteotomy for tibial valgus, and one patient underwent tibia lengthening; whereas in Group B, two patients needed corrective osteotomes for residual malaligments. CONCLUSION: Our study demonstrates that the new comprehensive protocol is extremely effective for achieving sound union in Congenital Pseudarthrosis of Tibia.

Gamma-Glutamyltransferase 1 Promotes Clear Cell Renal Cell Carcinoma Initiation and Progression.

Clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer. While the localized form of this disease can be treated surgically, advanced and metastatic stages are resistant to chemotherapies. Although more innovative treatments, such as targeted or immune-based therapies, exist, the need for new therapeutic options remains. ccRCC presents unique metabolic signatures and multiple studies have reported a significant increase in levels of reduced glutathione (GSH) and its precursors in ccRCC tumor samples compared with normal kidney tissues. These observations led us to investigate the effects of blocking the GSH pathway, particularly the gamma-glutamyltransferase 1 (GGT1) enzyme, in multiple ccRCC cell lines. In this study, we provide in vitro and in vivo evidence that GGT1/GSH pathway inhibition impacts ccRCC cell growth, through increased cell-cycle arrest. Of note, GGT1 inhibition also impairs ccRCC cell migration. Finally, pharmacologic GSH pathway inhibition decreases ccRCC cell proliferation and increases sensitivity to standard chemotherapy. Our results suggest that GGT1/GSH pathway inhibition represents a new strategy to overcome ccRCC chemoresistance. IMPLICATIONS: GGT1/GSH pathway inhibition represents a promising therapeutic strategy to overcome chemoresistance and inhibit progression of ccRCC tumors.

Glutathione metabolism in cancer progression and treatment resistance.

Glutathione (GSH) is the most abundant antioxidant found in living organisms and has multiple functions, most of which maintain cellular redox homeostasis. GSH preserves sufficient levels of cysteine and detoxifies xenobiotics while also conferring therapeutic resistance to cancer cells. However, GSH metabolism plays both beneficial and pathogenic roles in a variety of malignancies. It is crucial to the removal and detoxification of carcinogens, and alterations in this pathway can have a profound effect on cell survival. Excess GSH promotes tumor progression, where elevated levels correlate with increased metastasis. In this review, we discuss recent studies that focus on deciphering the role of GSH in tumor initiation and progression as well as mechanisms underlying how GSH imparts treatment resistance to growing cancers. Targeting GSH synthesis/utilization therefore represents a potential means of rendering tumor cells more susceptible to different treatment options such as chemotherapy and radiotherapy.

PPARγ is dispensable for clear cell renal cell carcinoma progression.

OBJECTIVE: Clear cell renal cell carcinoma (ccRCC) is a subtype of kidney cancer defined by robust lipid accumulation, which prior studies have indicated plays an important role in tumor progression. We hypothesized that the peroxisome proliferator-activated receptor gamma (PPARγ), detected in both ccRCC tumors and cell lines, promotes lipid storage in ccRCC and contributes to tumorigenesis in this setting. PPARγ transcriptionally regulates a number of genes involved in lipid and glucose metabolism in adipocytes, yet its role in ccRCC has not been described. The objective of this study was to elucidate endogenous PPARγ function in ccRCC cells. METHODS AND RESULTS: Using chromatin immunoprecipitation followed by deep sequencing (ChIP-seq), we found that PPARγ and its heterodimer RXR occupy the canonical DR1 PPAR binding motif at approximately 1000 locations throughout the genome that can be subdivided into adipose-shared and ccRCC-specific sites. CRISPR-Cas9 mediated, loss-of-function studies determined that PPARγ is dispensable for viability, proliferation, and migration of ccRCC cells in vitro and in vivo. Also, surprisingly, PPARγ deletion had little effect on the robust lipid accumulation that typifies the "clear cell" phenotype of kidney cancer. CONCLUSION: Our results suggest that PPARγ plays neither a tumor suppressive nor oncogenic role in advanced ccRCC, and thus single-agent therapeutics targeting PPARγ are unlikely to be effective for the treatment of this disease. The unique cistrome of PPARγ in ccRCC cells demonstrates the importance of cell type in determining the functions of PPARγ.

A Tyrosine Residue Along with a Glutamic Acid of the Omega-Like Loop Governs the Beta-Lactamase Activity of MSMEG_4455 in Mycobacterium smegmatis.

Mycobacterial beta-lactamases are involved in exerting beta-lactam resistance, though many of these proteins remain uncharacterized. Here, we have characterized MSMEG_4455 of Mycobacterium smegmatis as a beta-lactamase using molecular, biochemical and mutational techniques. To elucidate its nature in vivo and in vitro, and to predict its structure-function relationship in silico analysis is done. The MSMEG_4455 is cloned and expressed ectopically in a beta-lactamase deficient Escherichia coli mutant to establish the in vivo beta-lactamase like nature via minimum inhibitory concentration (MIC) determination. Likewise the in vivo results, purified soluble form of MSMEG_4455 showed beta-lactam hydrolysis pattern similar to group 2a penicillinase. In silico analyses of MSMEG_4455 reveal glutamic acid (E)193 and tyrosine (Y)194 of omega-like loop might have importance in strengthening hydrogen bond network around the active-site, though involvement of tyrosine is rare for beta-lactamase activity. Accordingly, these residues are mutated to alanine (A) and phenylalanine (F), respectively. The mutated proteins have partially lost their ability to exert beta-lactamase activity both in vivo and in vitro. The Y194F mutation had more prominent effect on the enzymatic activity. Therefore, we infer that Y194 is the key for beta-lactamase activity of MSMEG_4455.