The unusual di-domain structure of Dunaliella salina glycerol-3-phosphate dehydrogenase enables direct conversion of dihydroxyacetone phosphate to glycerol.

Dunaliella has been extensively studied due to its intriguing adaptation to high salinity. Its di-domain glycerol-3-phosphate dehydrogenase (GPDH) isoform is likely to underlie the rapid production of the osmoprotectant glycerol. Here, we report the structure of the chimeric Dunaliella salina GPDH (DsGPDH) protein featuring a phosphoserine phosphatase-like domain fused to the canonical glycerol-3-phosphate (G3P) dehydrogenase domain. Biochemical assays confirm that DsGPDH can convert dihydroxyacetone phosphate (DHAP) directly to glycerol, whereas a separate phosphatase protein is required for this conversion process in most organisms. The structure of DsGPDH in complex with its substrate DHAP and co-factor nicotinamide adenine dinucleotide (NAD) allows the identification of the residues that form the active sites. Furthermore, the structure reveals an intriguing homotetramer form that likely contributes to the rapid biosynthesis of glycerol.

Ribosome protection by antibiotic resistance ATP-binding cassette protein.

The ribosome is one of the richest targets for antibiotics. Unfortunately, antibiotic resistance is an urgent issue in clinical practice. Several ATP-binding cassette family proteins confer resistance to ribosome-targeting antibiotics through a yet unknown mechanism. Among them, MsrE has been implicated in macrolide resistance. Here, we report the cryo-EM structure of ATP form MsrE bound to the ribosome. Unlike previously characterized ribosomal protection proteins, MsrE is shown to bind to ribosomal exit site. Our structure reveals that the domain linker forms a unique needle-like arrangement with two crossed helices connected by an extended loop projecting into the peptidyl-transferase center and the nascent peptide exit tunnel, where numerous antibiotics bind. In combination with biochemical assays, our structure provides insight into how MsrE binding leads to conformational changes, which results in the release of the drug. This mechanism appears to be universal for the ABC-F type ribosome protection proteins.

Structure of BipA in GTP form bound to the ratcheted ribosome.

BPI-inducible protein A (BipA) is a member of the family of ribosome-dependent translational GTPase (trGTPase) factors along with elongation factors G and 4 (EF-G and EF4). Despite being highly conserved in bacteria and playing a critical role in coordinating cellular responses to environmental changes, its structures (isolated and ribosome bound) remain elusive. Here, we present the crystal structures of apo form and GTP analog, GDP, and guanosine-3',5'-bisdiphosphate (ppGpp)-bound BipA. In addition to having a distinctive domain arrangement, the C-terminal domain of BipA has a unique fold. Furthermore, we report the cryo-electron microscopy structure of BipA bound to the ribosome in its active GTP form and elucidate the unique structural attributes of BipA interactions with the ribosome and A-site tRNA in the light of its possible function in regulating translation.

Structure of the GTP Form of Elongation Factor 4 (EF4) Bound to the Ribosome.

Elongation factor 4 (EF4) is a member of the family of ribosome-dependent translational GTPase factors, along with elongation factor G and BPI-inducible protein A. Although EF4 is highly conserved in bacterial, mitochondrial, and chloroplast genomes, its exact biological function remains controversial. Here we present the cryo-EM reconstitution of the GTP form of EF4 bound to the ribosome with P and E site tRNAs at 3.8-Å resolution. Interestingly, our structure reveals an unrotated ribosome rather than a clockwise-rotated ribosome, as observed in the presence of EF4-GDP and P site tRNA. In addition, we also observed a counterclockwise-rotated form of the above complex at 5.7-Å resolution. Taken together, our results shed light on the interactions formed between EF4, the ribosome, and the P site tRNA and illuminate the GTPase activation mechanism at previously unresolved detail.

Similarity and diversity of translational GTPase factors EF-G, EF4, and BipA: From structure to function.

EF-G, EF4, and BipA are members of the translation factor family of GTPases with a common ribosome binding mode and GTPase activation mechanism. However, topological variations of shared as well as unique domains ensure different roles played by these proteins during translation. Recent X-ray crystallography and cryo-electron microscopy studies have revealed the structural basis for the involvement of EF-G domain IV in securing the movement of tRNAs and mRNA during translocation as well as revealing how the unique C-terminal domains of EF4 and BipA interact with the ribosome and tRNAs contributing to the regulation of translation under certain conditions. EF-G, EF-4, and BipA are intriguing examples of structural variations on a common theme that results in diverse behavior and function. Structural studies of translational GTPase factors have been greatly facilitated by the use of antibiotics, which have revealed their mechanism of action.

BswR controls bacterial motility and biofilm formation in Pseudomonas aeruginosa through modulation of the small RNA rsmZ.

Pseudomonas aeruginosa relies on cell motility and ability to form biofilms to establish infections; however, the mechanism of regulation remains obscure. Here we report that BswR, a xenobiotic response element-type transcriptional regulator, plays a critical role in regulation of bacterial motility and biofilm formation in P. aeruginosa. Transcriptomic and biochemical analyses showed that BswR counteracts the repressor activity of MvaT, controls the transcription of small RNA rsmZ and regulates the biogenesis of bacterial flagella. The crystal structure of BswR was determined at 2.3 Å resolution; the monomer comprises a DNA-binding domain with a helix-turn-helix motif in the N terminus and two helices (α6 and α7) with a V-shaped arrangement in the C-terminus. In addition to the contacts between the parallel helices α5 of two monomers, the two helical extensions (α6 and α7) intertwine together to form a homodimer, which is the biological function unit. Based on the result of DNase I protection assay together with structural analysis of BswR homodimer, we proposed a BswR-DNA model, which suggests a molecular mechanism with which BswR could interact with DNA. Taken together, our results unveiled a novel regulatory mechanism, in which BswR controls the motility and biofilm formation of P. aeruginosa by modulating the transcription of small RNA rsmZ.

Lichenoid Features and Fibrosis: Coexistence in Quid-induced Oral Lesions.

BACKGROUND: Oral submucous fibrosis (OSF) and quid associated oral lichenoid lesions (QOLL) are caused because of areca nut chewing and both show very characteristic histological changes. The present study aims to determine the histopathological presence of lichenoid features in cases of OSF as well as to determine the presence of fibrosis in cases of QOLL. MATERIALS AND METHODS: The study involved the retrospective analysis of hematoxylin and eosin stained slides of OSF (n = 50) and QOLL (n = 16). RESULTS: Seven cases of OSF revealed histopathological features of lichenoid reaction and four cases of QOLL revealed the presence of fibrosis in the underlying connective tissue. CONCLUSION: This study put forths a new finding that is, the presence of concomitant disease processes seen in the same patient at a histological level. It thus, stresses the need to evaluate all cases of OSF and QOLL for additional features which may be induced by areca nut chewing.

Fungal alkaloid malbrancheamide reorients the lipid binding domain of GRK5.

G protein-coupled receptors (GPCRs) are the largest group of receptors involved in various types of signaling. GPCR signaling is regulated via receptor phosphorylation by G protein-coupled receptor kinases 5 (GRK5). Calmodulin (CaM), a universal Ca2+ sensor, inhibits receptor phosphorylation by binding to GRK5. However, the inhibitor malbrancheamide (MBC), which binds at CaM C-lobe, allows for receptor phosphorylation. To understand the phosphorylation mechanism by GRK5, we carried out a MD simulation of the CaM/GRK5 complex in the presence and absence of the MBC inhibitor. The lipid binding domain (LBD) of GRK5 adopted different positions in the presence and absence of inhibitor. Furthermore, the inhibitor MBC restricted the movement of the N-lobe tether (NLT) loop, probably blocking the autophosphorylation of GRK5.Communicated by Ramaswamy H. Sarma.

Protein-Protein Interaction Studies Using Molecular Dynamics Simulation.

Protein-protein interaction (PPI) is a crucial event for many biological functions. Studying the molecular details of PPI requires structure determination using X-ray crystallography, nuclear magnetic resistance (NMR), and single particle Cryo-EM. However, sometimes it is not easy to solve the complex structure for various reasons. For example, complex may be unstable, not enough protein expression for structural studies, etc. Further, PPI are intricate processes, and its molecular details cannot be fully explained by experimental observations. Here, we describe a quick and simple method to study the PPI using the combinatorial approach of molecular dynamics simulation and biophysical methods.

Cytological assessment of healing palatal donor site wounds and grafted gingival wounds after application of ozonated oil: an eighteen-month randomized controlled clinical trial.

OBJECTIVE: The present study was undertaken to assess the therapeutic effects of topical ozonated oil on early healing of free gingival graft surgical sites. STUDY DESIGN: Twenty subjects were entered into this triple-blinded, randomized, placebo-controlled clinical trial, designed to evaluate the efficacy of ozonated oil on free gingival graft surgical wounds. Subjects were assigned to either the ozone group, in which ozonated oil was applied to the surgical wound, or the control group, in which non-ozonated oil was used as a control. Patients were postoperatively evaluated by cytological analysis. Cytological analysis consisted of the keratinisation and superficial cell indices measured at baseline, after 24 h, on the 3rd, 7th, 14th and 21st day and 2, 3, 8 and 18 months postoperatively. RESULTS: Cytological results showed that there was a significant (p < 0.001) improvement in epithelial healing by the 7th, 14th and 21st day and 2, 3 and 8 months postoperatively in the ozone group compared to the control group. CONCLUSION: The present study showed significant improvement in epithelial healing and gingival health after topical application of ozone-treated plant oil to gingival surgical sites.

Role of the Maxillofacial Surgeon in Identifying the Correlation Between Facial Bone Fractures and Traumatic Brain Injury - A Prospective Study.

Introduction: Maxillofacial trauma accounts for a high percentage of patients reporting to the emergency medicine department and being admitted in the hospital. The purpose of this study was to form a direct association between maxillofacial fractures and traumatic brain injury (TBI). Methods: Ninety patients with maxillofacial fractures that reported to/were referred to the Department of Oral and Maxillofacial Surgery were observed for features indicative of TBI based on clinical presentation and radiological interpretations. Parameters such as loss of consciousness, vomiting, dizziness, headache, seizures and the requirement for intubation, cerebrospinal fluid rhinorrhoea and otorrhoea were also assessed. Appropriate radiographs for the diagnosis of the fracture were taken followed by a computed tomography (CT) scan when indicated in accordance to the Canadian CT Head Rule. These scans were then assessed for contusion, extradural haemorrhage, subdural haemorrhage, subarachnoid haemorrhage, pneumocephalus and cranial bone fracture. Results: A total of 90 patients were evaluated, of which 91.1% were males and 8.9% were females. Association between the occurrence of head injury and different maxillofacial bone fractures using the Chi-square test showed a statistical significance of <0.001 in patients with naso-orbito-ethmoid as well as frontal bone fractures. There was a clear association between fractures present in the upper as well as the middle third of the face and traumatic head injury (P ≤ 0.001). Discussion: Patients with the frontal bone and zygomatic bone fractures have a high prevalence of TBI. Patients with the upper and middle third of the face injury are more prone to traumatic head injury and importance should be given to patients with the same and prevent poor prognosis.

A potential peptide inhibitor of SARS-CoV-2 S and human ACE2 complex.

The disease COVID-19 has caused heavy socio-economic burden and there is immediate need to control it. The disease is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. The viral entry into human cell depends on the attachment of spike (S) protein via its receptor binding domain (RBD) to human cell receptor angiotensin-converting enzyme 2 (hACE2). Thus, blocking the virus attachment to hACE2 could serve as potential therapeutics for viral infection. We have designed a peptide inhibitor (ΔABP-α2) targeting the RBD of S protein using in-silico approach. Docking studies and computed affinities suggested that peptide inhibitor binds at the RBD with ∼95-fold higher affinity than hACE2. Molecular dynamics (MD) simulation confirms the stable binding of inhibitor to hACE2. Immunoinformatics studies suggest non-immunogenic and non-toxic nature of peptide. Thus, the proposed peptide could serve as potential blocker for viral attachment.Communicated by Ramaswamy H. Sarma.

Infiltrating lipomatosis, an etiology for TMJ ankylosis?

Infiltrating lipomatosis is a rare benign condition causing diffuse fatty infiltration into the surrounding soft tissue and in rare cases causes hyperplasia of the adjacent bone. We report a case with clinical and radiological evidence of a 34-year-old female patient who reported a swelling in the left middle third of the face with exophytic temporomandibular joint (TMJ) ankylosis of the left side resulting in restricted mouth opening and facial asymmetry since 21 years. The number of cases reported in the literature is rare. Surgery is the treatment of choice.

Midface fracture pattern in a tertiary care hospital - A prospective study.

Introduction: The midface with multiple bones and cavities is vulnerable to fractures more commonly. Midface is one of the most frequently injured areas of the body, accounting for 23%-97% of all facial fractures. The classic LeFort type of fractures are uncommon nowadays and a more common picture is more severe forms primarily due to the high-speed vehicles that are a major cause of these fractures. It, therefore, has become imperative to determine the commonly occurring patterns of fractures in this area in the present time for a better insight into diagnosis and treatment plans. The aim of this article was to determine fracture patterns in midface trauma to ease the treatment planning in such a scenario. Materials and Methods: This prospective study included 114 patients who reported to a tertiary hospital during a 4-year period and were diagnosed with suspected midface fractures. The etiology and pattern of fractures of midface were assessed based on history, clinical examination, and imaging data. The diagnosis of a fracture was based on the clinical history, signs and symptoms, manual examination, and correct interpretation of radiographs and computed tomography. Midface fractures were recorded as LeFort I, II, III, dentoalveolar, palatal, zygomatic complex fracture, nasal bones, naso-orbital-ethmoidal complex, and orbital and zygomatic arch fractures. Etiological factors were classified as road traffic accidents (RTAs), fall, assault, and sports injuries. Results: During the 4-year period, a total of 114 patients were included. Patients' ages ranged from 17 to 68 years, with 102 males and 12 females. The most common fracture in this study was found to be zygomatic complex fractures (52%), and RTA was identified as the main cause of fracture in this study (79.2%). Conclusion: The midface fractures are more common in males due to the propensity of males to use two-wheelers more than females. The prominence of the zygoma makes it more vulnerable to fractures than rest of the bones in the midface. Increased speed of vehicles and lack of discipline in following traffic rules have resulted in RTA, being the biggest etiological factor in midface injuries.

Ophthalmic Complications Associated with Zygomatic Complex Fractures: A Randomised Descriptive Clinical Study.

Background and Objectives: Trauma to the maxillofacial region is usually associated with varying degrees of disruption of the soft and hard tissues in the region and injuries to the neighbouring structures such as eyes, brain, nasal apparatus and paranasal sinuses. Injuries to the middle third of the face commonly destroys the integrity of the orbital skeleton, and are frequently complicated by injury to the eye, ranging between 2.7 and 67% as reported in the literature. These injuries may result in loss of vision or compromised ocular function. When these injuries are severe, they may be detected with ease by any medical or maxillofacial surgeon but many injuries appear minimal and may be missed by the non-ophthalmologist. Methods: A total of thirty patients were selected who were diagnosed with a zygomatic complex fracture, irrespective of sex predilection and in the age group of 18-70 years. All patients underwent a thorough ophthalmic examination by an Oral and Maxillofacial Surgeon and an Ophthalmologist preoperatively and on postoperative day 2, and 7 and all findings were documented separately by the surgeon and the ophthalmologist and the findings were later compared. Results: The statistical analysis was performed using SPSS VERSION 21.0, Shapiro-Wilk test which was used to assess the normality. Descriptive analysis was done for age and gender distribution of study subjects, which are expressed as number and frequency. Cochran's q test was used to determine if there are differences in various study factors among OMFS and ophthalmologists at three time intervals which are expressed as number and frequency. Kappa agreement was used to assess the measurement of agreement between OMFS and ophthalmologists for each factor at each time interval and these are expressed as number and frequency, and p ≤ 0.05 is considered as statistically significant. Upon examination by an OMFS periorbital oedema (p = 0.000), periorbital ecchymosis (p = 0.002), chemosis (p = 0.02) and exophthalmos (p = 0.03) were considered clinically significant. Upon examination by an ophthalmologist subconjunctival haemorrhage (p = 0.05), periorbital oedema (p = 0.05), periorbital ecchymosis (p = 0.00), ptosis (p = 0.006), enopthalmos (p = 0.05) and diplopia (p = 0.05) were considered to be clinically significant. Upon correlation of the findings of the surgeon and the ophthalmologist it was seen that certain parameters like corneal injury, Phthisis bulbing, examination of posterior segment can be better done by the ophthalmologist. Interpretation and Conclusion: In conclusion, it was determined that there is a significant correlation between ophthalmic injuries caused by zygomatic complex fractures. There are a wide array of injuries affecting the eye due to trauma to the middle third of the face, and the superficial injury can be well evaluated and documented by a surgeon; however due to the extensive nature of the injuries in trauma and the importance of the eye, an ophthalmic examination cannot be ruled out. The need for evaluation of such injures deem it pertinent for observation by an ophthalmologist.

Taxifolin and Lucidin as Potential E6 Protein Inhibitors: p53 Function Re-Establishment and Apoptosis Induction in Cervical Cancer Cells.

Cervical cancer is the fourth leading cause of death in women worldwide, with 99% of cases associated with a human papillomavirus (HPV) infection. Given that HPV prophylactic vaccines do not exert a therapeutic effect in individuals previously infected, have low coverage of all HPV types, and have poor accessibility in developing countries, it is unlikely that HPV-associated cancers will be eradicated in the coming years. Therefore, there is an emerging need for the development of anti-HPV drugs. Considering HPV E6's oncogenic role, this protein has been proposed as a relevant target for cancer treatment. In the present work, we employed in silico tools to discover potential E6 inhibitors, as well as biochemical and cellular assays to understand the action of selected compounds in HPV-positive cells (Caski and HeLa) vs. HPV-negative (C33A) and non-carcinogenic (NHEK) cell lines. In fact, by molecular docking and molecular dynamics simulations, we found three phenolic compounds able to dock in the E6AP binding pocket of the E6 protein. In particular, lucidin and taxifolin were able to inhibit E6-mediated p53 degradation, selectively reduce the viability, and induce apoptosis in HPV-positive cells. Altogether, our data can be relevant for discovering promising leads for the development of specific anti-HPV drugs.

Structural characterization of BVU_3255, a methyltransferase from human intestine antibiotic resistant pathogen Bacteroides vulgatus.

Methylation is important for various cellular activities. To date, there is no report of any methyltransferase structure from the human intestine antibiotic resistant pathogen Bacteroides vulgatus. The protein BVU_3255 from B. vulgatus ATCC 8482 belongs to a SAM-dependent methyltransferase. Here, we report the crystal structure of apo BVU_3255, and its complexes with SAM and SAH, which revealed a typical class I Rossmann Fold Methyltransferase. Isothermal titration calorimetric studies showed that both SAM and SAH bind with equal affinity. The structural and sequence analysis suggested that BVU_3255 is a small molecule methyltransferase and involved in methylating the intermediates in ubiquinone biosynthesis pathway.

Purification, crystallization and diffraction studies of the methyltransferases BT_2972 and BVU_3255 from antibiotic-resistant pathogens of the genus Bacteroides from the human intestine.

The methyltransferases BT_2972 and BVU_3255 from two different Bacteroides species that are antibiotic-resistant pathogens from the human intestine were cloned, overexpressed and purified, yielding approximately 120 mg of each protein from 1 l culture. Apo BT_2972 and BVU_3255 and their complexes with S-adenosylmethionine or S-adenosylhomocysteine were crystallized in four different crystal forms using the hanging-drop vapour-diffusion method. These crystals diffracted to resolutions ranging from 2.8 to 2.2 Å. Sequence analysis suggested that the two proteins are homologous small-molecule methyltransferases.

The mechanism of complex formation between calmodulin and voltage gated calcium channels revealed by molecular dynamics.

Calmodulin, a ubiquitous eukaryotic calcium sensor responsible for the regulation of many fundamental cellular processes, is a highly flexible protein and exhibits an unusually wide range of conformations. Furthermore, CaM is known to interact with more than 300 cellular targets. Molecular dynamics (MD) simulation trajectories suggest that EF-hand loops show different magnitudes of flexibility. Therefore, the four EF-hand motifs have different affinities for Ca2+ ions, which enables CaM to function on wide range of Ca2+ ion concentrations. EF-hand loops are 2-3 times more flexible in apo CaM whereas least flexible in Ca2+/CaM-IQ motif complexes. We report a unique intermediate conformation of Ca2+/CaM while transitioning from extended to compact form. We also report the complex formation process between Ca2+/CaM and IQ CaM-binding motifs. Our results showed how IQ motif recognise its binding site on the CaM and how CaM transforms from extended to compact form upon binding to IQ motif.