Reversal of carbapenem resistance in Pseudomonas aeruginosa by camelid single domain antibody fragment (VHH) against the C4-dicarboxylate transporter.

Antimicrobial resistance is emerging as the new healthcare crisis necessitating the development of newer classes of drugs using non-traditional approaches. Pseudomonas aeruginosa, one of the most common pathogens involved in nosocomial infections, is extremely difficult to treat even with the last resort frontline drug, the carbapenems. As the pathogen has the ability to acquire resistance to new small-molecule antibiotics, being deployed, a novel biological approach has been tried using antibody fragments in combination with carbapenems and ß-lactams as adjunct therapy for an enduring solution to the problem. In this study, we developed a camelid antibody fragment (VHH) library against P. aeruginosa and isolated a highly potent hit, PsC23. Mass spectrometry identified the target to be a component of the C4-dicarboxylate transporter that feeds metabolites to the glyoxylate shunt particularly under conditions of oxidative stress. PsC23 is bacteriostatic at a concentration of 1.66 µM (25 µg ml-1) and shows a synergistic effect with both the classes of drugs at an effective concentration of 100-200 nM (1.5-3.0 µg ml-1) when co administered with them. In combination with meropenem the VHH completely cleared the infection from a neutropenic mouse with a carbapenem-resistant P. aeruginosa systemic infection. Blocking the glyoxylate shunt by PsC23 resulted in disruption of energy transduction due to a respiratory shift to the oxygen-depleted TCA cycle causing inhibition of efflux and increased free radical generation from carbapenems and ß-lactams exerting a strong bactericidal effect that reversed the resistance to multiple unrelated drugs.

Angiomyolipoma With Epithelial Cysts: A Rare but Distinct Variant of Angiomyolipoma.

Renal angiomyolipomas, common benign tumors, can exhibit slow growth in sporadic cases or have aggressive tendencies when linked to genetic conditions like tuberous sclerosis. This case report focuses on the exceptionally rare angiomyolipoma with epithelial cysts (AMLEC) variant, particularly challenging to diagnose due to its scarcity. Describing a 41-year-old woman's case, initially suspected to be renal cell carcinoma during an infertility evaluation, subsequent partial nephrectomy revealed a tumor comprising smooth muscle, blood vessels, and fat, with cystic regions featuring cuboidal linings and a layer devoid of abnormal cell activity. Immunohistochemistry confirmed specific markers within different tumor components, highlighting the diagnostic complexities of AMLEC and emphasizing the crucial role of histopathological examinations in accurate characterizations.

Treatment challenges in the management of difficult-to-treat gram-positive infections: A consensus view apropos therapeutic role of novel anti-MRSA antibiotics, levonadifloxacin (IV) and alalevonadifloxacin (oral).

PURPOSE: Treatment of antibiotic-resistant Gram-positive infections (GPIs), including methicillin-resistant Staphylococcus aureus (MRSA) is becoming increasingly difficult, particularly in patients with multiple co-morbidities who require antibiotics with greater safety and a consistent pharmacokinetic/pharmacodynamic (PK/PD) profile. Such difficult-to-treat GPIs are often associated with poor outcomes, extended hospital stay and increased expenditure. This can be partly attributed to the limited safety and aberrant PK/PD profile of existing anti-MRSA antibiotics. In this context, intravenous levonadifloxacin and its oral prodrug alalevonadifloxacin are novel anti-MRSA antibiotics that have significant advantages over conventional anti-Gram-positive antibiotics. The purpose of this paper was to generate a consensus on the optimal use of levonadifloxacin and alalevonadifloxacin for tackling resistant Gram-positive infections in patients with multiple co-morbidities. METHOD: Using a modified Delphi approach that combines critical appraisal of evidence and expert opinion, therapeutic use of levonadifloxacin and alalevonadifloxacin in various clinical scenarios and specific unmet conditions was deliberated. Fifteen expert members from medicine, critical-care, emergency, microbiology, and intensive-care disciplines participated and voted on 11 pre-conceived statements. When there was at least 70 % agreement, a consensus was reached. RESULTS: Following the voting, agreements were reached on 10 out of the 11 statements. Broadly, a consensus was reached in defining the therapeutic role of levonadifloxacin and alalevonadifloxacin in the treatment of various clinical indications involving resistant Gram-positive pathogens, including MRSA, in patients with co-morbidities, such as co-existing or increased risk for kidney dysfunction or hepatic disease and/or immunosuppression; also, in therapeutically challenging conditions caused by Gram-positive bacteria such as bacteraemia, bone and joint infection, diabetic foot infection, febrile neutropenia, and hospital-acquired pneumonia. CONCLUSIONS: This consensus supports the therapeutic use of levonadifloxacin and alalevonadifloxacin in the treatment of antibiotic-resistant GPIs, including those caused by MRSA and certain polymicrobial infections, in patients with multiple co-morbidities requiring drug with adequate safety and consistent efficacy.

Mechanism and evolutionary origins of alanine-tail C-degron recognition by E3 ligases Pirh2 and CRL2-KLHDC10.

In ribosome-associated quality control (RQC), nascent polypeptides produced by interrupted translation are modified with C-terminal polyalanine tails ("Ala-tails") that function outside ribosomes to induce ubiquitylation by E3 ligases Pirh2 (p53-induced RING-H2 domain-containing) or CRL2 (Cullin-2 RING ligase2)-KLHDC10. Here, we investigate the molecular basis of Ala-tail function using biochemical and in silico approaches. We show that Pirh2 and KLHDC10 directly bind to Ala-tails and that structural predictions identify candidate Ala-tail-binding sites, which we experimentally validate. The degron-binding pockets and specific pocket residues implicated in Ala-tail recognition are conserved among Pirh2 and KLHDC10 homologs, suggesting that an important function of these ligases across eukaryotes is in targeting Ala-tailed substrates. Moreover, we establish that the two Ala-tail-binding pockets have convergently evolved, either from an ancient module of bacterial provenance (Pirh2) or via tinkering of a widespread C-degron-recognition element (KLHDC10). These results shed light on the recognition of a simple degron sequence and the evolution of Ala-tail proteolytic signaling.

Mechanism and evolutionary origins of Alanine-tail C-degron recognition by E3 ligases Pirh2 and CRL2-KLHDC10.

In Ribosome-associated Quality Control (RQC), nascent-polypeptides produced by interrupted translation are modified with C-terminal polyalanine tails ('Ala-tails') that function outside ribosomes to induce ubiquitylation by Pirh2 or CRL2-KLHDC10 E3 ligases. Here we investigate the molecular basis of Ala-tail function using biochemical and in silico approaches. We show that Pirh2 and KLHDC10 directly bind to Ala-tails, and structural predictions identify candidate Ala-tail binding sites, which we experimentally validate. The degron-binding pockets and specific pocket residues implicated in Ala-tail recognition are conserved among Pirh2 and KLHDC10 homologs, suggesting that an important function of these ligases across eukaryotes is in targeting Ala-tailed substrates. Moreover, we establish that the two Ala-tail binding pockets have convergently evolved, either from an ancient module of bacterial provenance (Pirh2) or via tinkering of a widespread C-degron recognition element (KLHDC10). These results shed light on the recognition of a simple degron sequence and the evolution of Ala-tail proteolytic signaling.

Preoperative Surgical Site Hair Removal for Elective Abdominal Surgery: Does It Have Impact on Surgical Site Infection.

Introduction Postoperative surgical site infection (SSI) forms the major burden of nosocomial infections in surgical patients. There is prevalent practice of surgical site hair shaving as a part of preoperative preparation. There is uncertainty regarding the benefit versus harm of shaving for SSIs. Hairs at surgical sites are removed prior to surgery most often by shaving. We performed this study to look for what impact preoperative hair removal by shaving has on postoperative SSI. Methods We performed prospective comparative cohort study in patients undergoing elective abdominal surgeries. We included clean and clean-contaminated surgeries in immunocompetent patients of which half were shaved and other half not shaved prior to surgery. Other confounding factors like skin cleaning, aseptic technique of surgery, antibiotic prophylaxis and treatment, and postoperative wound care were as per care. Patients were assessed for presence and grade of SSI postoperatively on day 7, 14, and 30. Results were analyzed statistically using chi-square and Fischer's exact tests for significance in entire sample as well as in demographic subgroups. Results Overall SSI rate was 11.42%. There was no statistically significant difference in SSI rates between patients who underwent preoperative surgical site hair removal by shaving (232) and who did not have shaving (232) on all the three different assessment timelines in postoperative period, namely, day 7, 14, and 30. Although the absolute number of patients who had SSI was more in those who underwent preoperative surgical site hair removal by shaving, the difference was not statistically significant ( p > 0.05). But on subgroup analysis patients with clean-contaminated surgeries ( p = 0.037) and patients with surgeries lasting for less than 2 hours (Fischer's exact = 0.034) had significantly higher SSI in the shaved group compared with unshaved on day 14. Conclusion As per our results, preoperative shaving did not significantly increase overall SSI except in subgroup of clean-contaminated surgeries and in surgeries of less than 2 hours' duration. So especially in these patients avoiding preoperative surgical site hair shaving may be used as one of the infection control measures.

Risk of Surgeon Contracting COVID-19 while Operating on COVID-19-Positive Patient, Impact of Safety Measures: Lessons Learnt.

Introduction On March 11, 2020, the novel coronavirus disease 2019 (COVID-19) was declared as a pandemic. General surgeons provide care to COVID-19 positive patients requiring emergency surgeries and hence are exposed to the virus. Surgery on COVID-19-positive patient itself is a major risk factor for surgeon to contract COVID-19 infection. Noticeably, there is no data regarding number of surgeons who have contracted COVID-19 after operating on COVID-19-positive patients. Hence, the aim of this study was to find out the exact incidence of COVID-19 among surgeons operating on COVID-19-positive patients and to analyze the impact of safety measures practiced by us. Methodology The study was conducted in a tertiary care center in Mumbai. It was a retrospective observational study with duration of 5 months from May 1, 2020, to September 30, 2020. Only those surgeons (faculty and resident doctors) were included who performed surgeries on COVID-19-positive patients (diagnosed by reverse-transcription polymerase chain reaction [RT-PCR] test) and gave consent for participation. As an institutional protocol, all patients undergoing surgery were tested by RT-PCR test (irrespective of chest X-ray or symptoms). Nasopharyngeal swabs for COVID-19 disease were collected prior to procedure but in some of these, results came after surgery. Still such patients were included in this study. Irrespective of COVID-19 status, same precautions were taken for all surgeries. The details of the patients like date of surgery, age, sex, surgery performed, duration of surgery, type of anesthesia used, and operating surgeon were noted from operation room (OR) register. Details of surgeons (faculty and resident doctors) who fulfilled inclusion criteria were noted by interview in terms of their demographic parameters, such as age, sex, designation, experience in years after completing postgraduation, comorbidities, whether they ever contracted COVID-19 (if yes, date), and safety measures practiced (yes, no, or cannot recollect). Patient was assumed to be the source only if the surgeon contracted COVID-19 within 14 days of surgery. Results A total of 34 surgeons (7 faculty and 27 residents) conducted 41 surgeries on COVID-19-positive patients during the study period. All of them gave consent for participation in the study. More than one surgeon was involved in a particular surgery. Hence, there were 78 occasions (faculty during 16 occasions and resident doctors on 62 occasions) when surgeons were at risk to contract COVID-19 while operating on patients ( n = 78). These surgeries had similar/comparable risk of COVID-19 exposure to surgeons and procedures with excessive exposure risk like airway procedures did not happen during the study period. The mean age of surgeon was 27.92 years ( n = 78, standard deviation = 5.71) and median experience of faculty after completion of postgraduate degree was 7 years ( n = 16, interquartile range [IQR] = 1.25-11.0). Only one faculty had comorbidity (diabetes mellitus). Duration of surgeries ranged from 50 to 420 minutes with median being 190 minutes ( n = 41, IQR = 120-240). Only one surgeon (male faculty) contracted COVID-19 within 14 days of surgery (1.3% incidence, n = 78), a total of seven surgeons contracted COVID-19 during study period but not within 14 days of surgery (source other than patient operated) and all remaining surgeons were asymptomatic throughout the study period. The surgeon who contracted COVID-19 (within 14 days) performed surgery for 260 minutes and under general anesthesia. All the surgeons followed standard steps of donning and doffing, used personal protective equipment (PPE) body cover, shoe cover, hood, double pair of gloves, and N-95 masks at all times ( n = 78). Intubation box was used in 100% cases of general anesthesia ( n = 19). Fogging of OR after each surgery and interval of 20 minutes between surgeries was followed in 100% cases. Also, patient was wearing mask at all possible times and anesthetist and support staff used PPE during all surgeries. Hence the relationship between COVID-19 status and these safety measures cannot be assessed. Goggles and face shields were not used on 88.5% ( n = 78) and 93.2% ( n = 73, because five surgeons could not recollect whether they used face shields or not) occasions, respectively. Also, immediate shower after surgery was not taken on 93.6% occasions ( n = 78). The surgeon who contracted COVID-19 had neither used goggles nor face shield. Also, he did not take shower immediately after surgery. However, there was no significant association between use of goggles, face shields, or shower after surgery and contraction of COVID-19 after operating patients (Fisher's exact p = 1.000). Air conditioner was switched-off only in 7.3% surgeries ( n = 41). Smoke evacuator (cautery with attached suction) was not used in 97.6% cases. Clinical documentation (handling of patient's files) was done outside OR in only 17.1% surgeries ( n = 41). However, there was no significant association between these safety measures and contraction of COVID-19 (Fisher's exact p = 1.000). General anesthesia was used in 19 surgeries (46.3%) while spinal anesthesia in 16 surgeries (39%), local anesthesia in 5 surgeries (12.2%), and total intravenous anesthesia (TIVA) in one surgery (2.4%). However, there was no significant association between type of anesthesia given during surgery and contraction of COVID-19 after operating on patients with Fisher's exact p -value of 1.000. Conclusion Even though safety measures, like goggles, face shield, switching-off of air conditioner, use of smoke evacuator, and shower, immediately after surgery were not practiced in majority of cases, surgeon positivity rate was significantly less. Also, there was no use of negative pressure in OR. Hence, their significance becomes questionable. Although adopting all universal safety measures is in everyone's best interest, it is seldom cost-effective. To reduce resource exhaustion, especially in a pandemic situation, the use of various safety measures and staff must be balanced. Use and promotion of unnecessary safety measures leads to added health care costs and fear among health care workers in case of unavailability. Even though our study has a small sample size and has its own limitations, it can guide future studies to strengthen recommendations and reduce health care costs. This will also help in future epidemics/pandemics.

Bacterial ribosome collision sensing by a MutS DNA repair ATPase paralogue.

Ribosome stalling during translation is detrimental to cellular fitness, but how this is sensed and elicits recycling of ribosomal subunits and quality control of associated mRNA and incomplete nascent chains is poorly understood1,2. Here we uncover Bacillus subtilis MutS2, a member of the conserved MutS family of ATPases that function in DNA mismatch repair3, as an unexpected ribosome-binding protein with an essential function in translational quality control. Cryo-electron microscopy analysis of affinity-purified native complexes shows that MutS2 functions in sensing collisions between stalled and translating ribosomes and suggests how ribosome collisions can serve as platforms to deploy downstream processes: MutS2 has an RNA endonuclease small MutS-related (SMR) domain, as well as an ATPase/clamp domain that is properly positioned to promote ribosomal subunit dissociation, which is a requirement both for ribosome recycling and for initiation of ribosome-associated protein quality control (RQC). Accordingly, MutS2 promotes nascent chain modification with alanine-tail degrons-an early step in RQC-in an ATPase domain-dependent manner. The relevance of these observations is underscored by evidence of strong co-occurrence of MutS2 and RQC genes across bacterial phyla. Overall, the findings demonstrate a deeply conserved role for ribosome collisions in mounting a complex response to the interruption of translation within open reading frames.

Robotic-assisted TKA leads to a better prosthesis alignment and a better joint line restoration as compared to conventional TKA: a prospective randomized controlled trial.

PURPOSE: Correct positioning and alignment of the prosthesis is a very important factor for durability of prosthesis and implant survival which is improved with the use of technology in total knee arthroplasty. However, the long-term functional outcomes and survivorship are unclear. For this study, it was hypothesized that mechanical axis alignment of lower limb, post-operative joint line restoration, femoral and tibial component alignment is more accurate with the new handheld semi-active robotic-assisted TKA. METHOD: From April-2019 to March-2020, 60 patients with unilateral knee osteoarthritis who underwent total knee arthroplasties were included in this prospective randomised controlled study. Computer generated randomization was used. Study included 48 female patients and 12 male patients. Pre-operative and post-operative radiographic measurements were done and compared between the two groups. RESULTS: There was a significant difference between two groups with respect to mechanical axis deviation, joint line deviation and coronal alignment of femoral and tibial prosthesis. Mechanical axis deviation > 3° was seen in eight cases (28.5%) in C-TKA group compared to one case (3.1%) in RA-TKA (p 0.019). Joint line deviation of 3.5 mm was noted in C-TKA group as compared to 0.9 mm in RA-TKA group (p < 0.001) which was statistically significant. However, whether this difference of 2.6 mm of joint line elevation between C-TKA and RA-TKA leads to any difference in clinical outcome in terms of knee kinematics and knee flexion needs to be investigated with further studies. Clinically restoring normal joint line is important for improved knee function after primary TKA. No significant difference was noted in femoral component rotation on post-operative computed tomography (CT) scan. CONCLUSION: The novel imageless, handheld semi-autonomous robotic system for TKA is highly accurate with respect to component positioning in coronal plane and mechanical alignment as compared to conventional TKA. Joint line is elevated in conventional TKA but is accurately restored using the robotic-assisted TKA which may lead to better patellofemoral kinematics. LEVEL OF EVIDENCE: I.

An open repository of real-time COVID-19 indicators.

The COVID-19 pandemic presented enormous data challenges in the United States. Policy makers, epidemiological modelers, and health researchers all require up-to-date data on the pandemic and relevant public behavior, ideally at fine spatial and temporal resolution. The COVIDcast API is our attempt to fill this need: Operational since April 2020, it provides open access to both traditional public health surveillance signals (cases, deaths, and hospitalizations) and many auxiliary indicators of COVID-19 activity, such as signals extracted from deidentified medical claims data, massive online surveys, cell phone mobility data, and internet search trends. These are available at a fine geographic resolution (mostly at the county level) and are updated daily. The COVIDcast API also tracks all revisions to historical data, allowing modelers to account for the frequent revisions and backfill that are common for many public health data sources. All of the data are available in a common format through the API and accompanying R and Python software packages. This paper describes the data sources and signals, and provides examples demonstrating that the auxiliary signals in the COVIDcast API present information relevant to tracking COVID activity, augmenting traditional public health reporting and empowering research and decision-making.

Real-World Experience of Immunotherapy from India in Recurrent Squamous Cell Carcinoma of Head and Neck Cancer.

Background Recurrent metastatic head and neck squamous cell carcinoma (HNSCC) patients carry a poor prognosis and have limited therapeutic options. In the randomized phase-3 trial CheckMate 141, nivolumab showed benefit in overall survival (OS) with manageable toxicity. Nivolumab is available for clinical practice since 2017 in India. The aim of this study is to evaluate the efficacy and safety of nivolumab in real-world settings in India. Materials and Methods This is a retrospective, single-center study on the use of nivolumab with advanced or metastatic HNSCC in India. Eligible patients had histologically confirmed, recurrent squamous cell carcinoma of the head and neck (including metastatic disease) of the oral cavity, pharynx, or larynx that was not amenable to curative treatment, tumor progression, or recurrence after the administration of platinum-containing chemotherapy administered as adjuvant therapy or in the context of primary or recurrent disease. We assessed demographics, safety (the Common Terminology Criteria for Adverse Events Version 4.0), response evaluation (the Response Evaluation Criteria in Solid Tumors Version 1.1), progression-free survival (PFS), and OS. Results Among patients with platinum-refractory, recurrent HNSCC, and treatment with nivolumab resulted in median PFS of 2 months and median OS of 5 months, which is inferior to what was seen in CheckMate 141. Fifteen of 20 patients (75%) had progressive disease, 3 (15%) showed a partial response, and 2 (10%) had stable disease. Conclusion Nivolumab was well tolerated in our study with fewer toxic effects, and an inferior median survival was reached as compared with CheckMate 141 in platinum refractory, recurrent HNSCC patients treated with nivolumab because 90% of patients in our study received nivolumab as second-line therapy after progression. Our study encourages the use of nivolumab in this population.

Real-World Experience in Toxicity with Bevacizumab in Indian Cancer Patients.

Background Bevacizumab, a humanized monoclonal antibody, known to block the binding of all known vascular endothelial growth factor-A isomers to their receptors, is used in solid cancers, especially in advanced settings where its role is proven to be stronger than localized stages. Furthermore, various studies have suggested that adding bevacizumab to first-line standard therapy in advanced solid cancers, such as colorectal cancer, lung cancer, ovarian cancer, renal cancer, and breast cancer, significantly prolongs progression-free survival, overall survival, and response rates. However, this ability is limited and variable in cancer subtype. The toxicity profile of bevacizumab is outspread, ranging from mild gastrointestinal side effects, proteinuria to life-threatening hemorrhagic tendency, ischemic thromboembolism, and intestinal perforation. However, it has never been studied in Indian subset of patients till date. Materials and Methods We performed an institutional retrospective study of 41 patients with a pathologically proven ovarian, colorectal, lung, mesothelioma, melanoma, round cell tumors, and GBMs who received bevacizumab (2.5 mg/kg/week [5 mg/kg every 2 weeks or 7.5 mg/kg every 3 weeks]) with or without chemotherapy, between January 2016 and January 2019 at our center in North India. Results Forty-one patients, including 12 (29) advanced ovarian cancer, 12 (29) colon cancer, 10 (24) rectal cancer, 1 (2) appendicular cancer, 1 (2) mesothelioma, 1 (2) melanoma, 1 (2) desmoplastic round cell tumor, and 3 (7) GBM, were treated with bevacizumab. The incidence of arterial thrombus and hemorrhage was 2 and 10%, respectively, whereas venous thrombus and fistula were not seen and not related to age. No fatal adverse event was recorded. The global incidence of severe (grade 3/4) arterial hypertension (HTN) was 49%. It was safely managed in all cases, and no grade 4 (life-threatening complication) occurred. The incidence of severe HTN was significantly higher in elderly patients than in younger ones (72 vs. 40%), proteinuria was found to be more frequent in the younger age group as compared with older age group (7 vs. 3%). Also to note, the incidence of congestive heart failure and subacute intestinal obstruction was found in 5% of patients, wherein all four patients belonged to the older subgroup. Furthermore, Grade 3 hypersensitivity reaction was found in one patient in the younger subgroup which warranted immediate termination of bevacizumab.

Convergence of mammalian RQC and C-end rule proteolytic pathways via alanine tailing.

Incompletely synthesized nascent chains obstructing large ribosomal subunits are targeted for degradation by ribosome-associated quality control (RQC). In bacterial RQC, RqcH marks the nascent chains with C-terminal alanine (Ala) tails that are directly recognized by proteasome-like proteases, whereas in eukaryotes, RqcH orthologs (Rqc2/NEMF [nuclear export mediator factor]) assist the Ltn1/Listerin E3 ligase in nascent chain ubiquitylation. Here, we study RQC-mediated proteolytic targeting of ribosome stalling products in mammalian cells. We show that mammalian NEMF has an additional, Listerin-independent proteolytic role, which, as in bacteria, is mediated by tRNA-Ala binding and Ala tailing. However, in mammalian cells Ala tails signal proteolysis indirectly, through a pathway that recognizes C-terminal degrons; we identify the CRL2KLHDC10 E3 ligase complex and the novel C-end rule E3, Pirh2/Rchy1, as bona fide RQC pathway components that directly bind to Ala-tailed ribosome stalling products and target them for degradation. As Listerin mutation causes neurodegeneration in mice, functionally redundant E3s may likewise be implicated in molecular mechanisms of neurodegeneration.

Purification and preliminary characterization of four Rel homologues from pathogenic bacteria: Implications for species-specific inhibitor design.

Resistance to antibiotics is a serious concern to treat infectious diseases and also, for food preservation. Existing antibiotics generally inhibit enzymes participating in key bacterial processes, such as formation of cell wall, replication, transcription and translation. However, bacteria have rapidly evolved new mechanisms to combat these antibiotics and it hence becomes indispensable to identify newer targets and identify/design inhibitors against them. Another concern is that most antibiotics are broad spectrum; they largely bind and inhibit the active site of the target enzyme. Rel proteins, which synthesize (and hydrolyze) (p)ppGpp in response to a variety of stress encountered by bacteria, is a profitable target owing to its distinct absence in humans and an intricate regulation of the catalytic activities. Inactivation of (p)ppGpp synthesis by Rel, disables bacterial survival in Mycobacterium tuberculosis and Staphylococcus aureus, while inactivating the hydrolysis activity was lethal. The poor MIC values of the currently known Rel inhibitors present a distinct opportunity to develop better inhibitors and warrants a detailed structural characterization and understanding of the complex regulation in Rel proteins. It will open new avenues for the design of effective, species-specific inhibitors. In an attempt to identify unique sites for inhibitor design using structure-based approaches, we initiate a study of Rel homologues from four different pathogenic bacteria, in order to compare their attributes with well characterized Rel homologues. Here, we present cloning, over-expression, purification and preliminary characterization of these four homologues; and suggest similarities and differences that can be exploited for inhibitor design.

A revised mechanism for (p)ppGpp synthesis by Rel proteins: The critical role of the 2'-OH of GTP.

Bacterial Rel proteins synthesize hyperphosphorylated guanosine nucleotides, denoted as (p)ppGpp, which by inhibiting energy requiring molecular pathways help bacteria to overcome the depletion of nutrients in its surroundings. (p)ppGpp synthesis by Rel involves transferring a pyrophosphate from ATP to the oxygen of 3'-OH of GTP/GDP. Initially, a conserved glutamate at the active site was believed to generate the nucleophile necessary to accomplish the reaction. Later this role was alluded to a Mg2+ ion. However, no study has unequivocally established a catalytic mechanism for (p)ppGpp synthesis. Here we present a revised mechanism, wherein for the first time we explore a role for 2'-OH of GTP and show how it is important in generating the nucleophile. Through a careful comparison of substrate-bound structures of Rel, we illustrate that the active site does not discriminate GTP from dGTP, for a substrate. Using biochemical studies, we demonstrate that both GTP and dGTP bind to Rel, but only GTP (but not dGTP) can form the product. Reactions performed using GTP analogs substituted with different chemical moieties at the 2' position suggest a clear role for 2'-OH in catalysis by providing an indispensable hydrogen bond; preliminary computational analysis further supports this view. This study elucidating a catalytic role for 2'-OH of GTP in (p)ppGpp synthesis allows us to propose different mechanistic possibilities by which it generates the nucleophile for the synthesis reaction. This study underscores the selection of ribose nucleotides as second messengers and finds its roots in the old RNA world hypothesis.

Discovery of Arginine Ethyl Ester as Polyglutamine Aggregation Inhibitor: Conformational Transitioning of Huntingtin N-Terminus Augments Aggregation Suppression.

Huntington's disease (HD) is a genetic disorder caused by a CAG expansion mutation in the huntingtin gene leading to polyglutamine (polyQ) expansion in the N-terminal part of huntingtin (Httex1). Expanded polyQ, through a complex aggregation pathway, forms aggregates in neurons and presents a potential therapeutic target. Here we show Httex1 aggregation suppression by arginine and arginine ethyl ester (AEE) in vitro, as well as in yeast and mammalian cell models of HD, bearing expanded polyQ. These molecules also rescue locomotion dysfunction in HD Drosophila model. Both molecules alter the hydrogen bonding network of polyQ to enhance its aqueous solubility and delay aggregation. AEE shows direct binding with the NT17 part of Httex1 to induce structural changes to impart an enhanced inhibitory effect. This study provides a platform for the development of better arginine based therapeutic molecules against polyQ-rich Httex1 aggregation.