YLR419W is the homolog of the mammalian translation initiation factor DHX29.

27 years after the yeast genome sequencing, the function of many ORFs remain unknown. Despite the evolutionary distance between human and yeast, homology with the conserved DEAH/DExH-box helicase domains allowed us to list DHX29, DHX36 and DHX57 as three putative homologs of the yeast Ylr419wp. Functional studies first linked the Ylr419w protein to the translating ribosome and cross-linking and analysis of cDNA (CRAC) experiments determined the precise region of Ylr419wp in contact with the ribosome. It corresponds to the loop of the h16 helix in the 18S rRNA designing the translation initiation factor DHX29, as the functional homolog of Ylr419wp.

Influence of Preoperative Sagittal Alignment on Functional Recovery in Operated Cases of Cervical Spondylotic Myelopathy.

Objective We examine the influence of preoperative cervical sagittal curvature (lordotic or nonlordotic) on the functional recovery of surgically managed cases of cervical spondylotic myelopathy (CSM). The impact of sagittal alignment on the functional improvement of operated CSM cases has not been thoroughly investigated. Materials and Methods We did retrospective analysis of consecutively operated cases of CSM from March 2019 to April 2021. Patients were grouped into two categories: lordotic curvature (with Cobb angle > 10 degrees) and nonlordotic curvature (including neutral [Cobb angle 0-10 degrees] and kyphotic [Cobb angle < 0 degrees]). Demographic data, and preoperative and postoperative functional outcome scores (modified Japanese Orthopaedic Association [mJOA] and Nurick grade) were analyzed for dependency on preoperative curvature, and correlations between outcomes and sagittal parameters were assessed. Results In the analysis of 124 cases, 63.1% (78 cases) were lordotic (mean Cobb angle of 23.57 ± 9.1 degrees; 11-50 degrees) and 36.9% (46 cases) were nonlordotic (mean Cobb angle of 0.89 ± 6.5 degrees; -11 to 10 degrees), 32 cases (24.6%) had neutral alignment, and 14 cases (12.3%) had kyphotic alignment. At the final follow-up, the mean change in mJOA score, Nurick grade, and functional recovery rate (mJOArr) were not significantly different between the lordotic and nonlordotic group. In the nonlordotic group, cases with anterior surgery had a significantly better mJOArr than those with posterior surgery ( p = 0.04), whereas there was similar improvement with either approach in lordotic cases. In the nonlordotic group, patients who gained lordosis (78.1%) had better recovery rates than those who had lost lordosis (21.9%). However, this difference was not statistically significant. Conclusion We report noninferiority of the functional outcome in the cases with preoperative nonlordotic alignment when compared with those with lordotic alignment. Further, nonlordotic patients who were approached anteriorly fared better than those approached posteriorly. Although increasing sagittal imbalance in nonlordotic spines portend toward higher preoperative disability, gain in lordosis in such cases may improve results. We recommend further studies with larger nonlordotic subjects to elucidate the impact of sagittal alignment on functional outcome.

Clinicoradiological Risk Factors Associated with Inability to Achieve Minimum Clinically Important Difference in Operated Cases of Cervical Spondylotic Myelopathy.

STUDY DESIGN: This is a retrospective cohort study. PURPOSE: This study aimed to identify the clinicoradiological risk factors associated with the inability to achieve minimum clinically important difference (MCID) on the modified Japanese Orthopaedic Association (mJOA) Scale in operated cases of cervical spondylotic myelopathy (CSM). OVERVIEW OF LITERATURE: Only a few studies have evaluated the outcomes of surgery performed for CSM using MCID on the mJOA scale. METHODS: We analyzed 124 operated CSM cases from March 2019 to April 2021 for preoperative clinical features, cervical sagittal radiographic parameters, and magnetic resonance imaging (MRI) signal intensities (SI). The risk factors associated with missing the MCID (poor outcome) on mJOA at the final follow-up were identified using binary logistic regression. Multivariate analysis was used to find significant risk factors, and odds ratios (OR) were computed. RESULTS: A total of 110 men (89.2%) and 14 women (10.8%) with an average age of 53.5±13.2 years were included in the analysis. During the last follow-up, 89 cases (72.1%) achieved MCID (meaningful gains following surgery) while 35 (27.9%) could not. The final model identified the following parameters as significant risk factors for poor outcome: increased duration of symptoms (OR, 6.77; p=0.001), lower preoperative mJOA scale (OR, 0.75; p=0.029), the presence of multilevel T2-weighted (T2W) MRI SI (OR, 4.79; p=0.004), and larger change in cervical sagittal vertical axis (ΔcSVA) (OR, 1.06; p=0.013). Also, an increase in cSVA postoperatively correlated with a reduced functional recovery rate (r=-0.4, p<0.001). CONCLUSIONS: Surgery for CSM leads to significant functional benefits. However, poorer outcomes are observed in cases of greater duration of symptoms, higher preoperative severity with multilevel T2W MRI SI, and a larger increase in the postoperative cSVA (sagittal imbalance).

In Silico Structure-Based Vaccine Design.

Structure-based vaccine design (SBVD) is an important technique in computational vaccine design that uses structural information on a targeted protein to design novel vaccine candidates. This increasing ability to rapidly model structural information on proteins and antibodies has provided the scientific community with many new vaccine targets and novel opportunities for future vaccine discovery. This chapter provides a comprehensive overview of the status of in silico SBVD and discusses the current challenges and limitations. Key strategies in the field of SBVD are exemplified by a case study on design of COVID-19 vaccines targeting SARS-CoV-2 spike protein.

Chromosomal Position of Ribosomal Protein Genes Affects Long-Term Evolution of Vibrio cholerae.

It is unclear how gene order within the chromosome influences genome evolution. Bacteria cluster transcription and translation genes close to the replication origin (oriC). In Vibrio cholerae, relocation of s10-spc-α locus (S10), the major locus of ribosomal protein genes, to ectopic genomic positions shows that its relative distance to the oriC correlates to a reduction in growth rate, fitness, and infectivity. To test the long-term impact of this trait, we evolved 12 populations of V. cholerae strains bearing S10 at an oriC-proximal or an oriC-distal location for 1,000 generations. During the first 250 generations, positive selection was the main force driving mutation. After 1,000 generations, we observed more nonadaptative mutations and hypermutator genotypes. Populations fixed inactivating mutations at many genes linked to virulence: flagellum, chemotaxis, biofilm, and quorum sensing. Throughout the experiment, all populations increased their growth rates. However, those bearing S10 close to oriC remained the fittest, indicating that suppressor mutations cannot compensate for the genomic position of the main ribosomal protein locus. Selection and sequencing of the fastest-growing clones allowed us to characterize mutations inactivating, among other sites, flagellum master regulators. Reintroduction of these mutations into the wild-type context led to a ≈10% growth improvement. In conclusion, the genomic location of ribosomal protein genes conditions the evolutionary trajectory of V. cholerae. While genomic content is highly plastic in prokaryotes, gene order is an underestimated factor that conditions cellular physiology and evolution. A lack of suppression enables artificial gene relocation as a tool for genetic circuit reprogramming. IMPORTANCE The bacterial chromosome harbors several entangled processes such as replication, transcription, DNA repair, and segregation. Replication begins bidirectionally at the replication origin (oriC) until the terminal region (ter) organizing the genome along the ori-ter axis gene order along this axis could link genome structure to cell physiology. Fast-growing bacteria cluster translation genes near oriC. In Vibrio cholerae, moving them away was feasible but at the cost of losing fitness and infectivity. Here, we evolved strains harboring ribosomal genes close or far from oriC. Growth rate differences persisted after 1,000 generations. No mutation was able to compensate for the growth defect, showing that ribosomal gene location conditions their evolutionary trajectory. Despite the high plasticity of bacterial genomes, evolution has sculpted gene order to optimize the ecological strategy of the microorganism. We observed growth rate improvement throughout the evolution experiment that occurred at expense of energetically costly processes such the flagellum biosynthesis and virulence-related functions. From the biotechnological point of view, manipulation of gene order enables altering bacterial growth with no escape events.

The Formation of Neochromosomes during Experimental Evolution in the Yeast Saccharomyces cerevisiae.

Novel, large-scale structural mutations were previously discovered during the cultivation of engineered Saccharomyces cerevisiae strains in which essential tRNA synthetase genes were replaced by their orthologs from the distantly related yeast Yarrowia lipolytica. Among those were internal segmental amplifications forming giant chromosomes as well as complex segmental rearrangements associated with massive amplifications at an unselected short locus. The formation of such novel structures, whose stability is high enough to propagate over multiple generations, involved short repeated sequences dispersed in the genome (as expected), but also novel junctions between unrelated sequences likely triggered by accidental template switching within replication forks. Using the same evolutionary protocol, we now describe yet another type of major structural mutation in the yeast genome, the formation of neochromosomes, with functional centromeres and telomeres, made of extra copies of very long chromosomal segments ligated together in novel arrangements. The novel junctions occurred between short repeated sequences dispersed in the genome. They first resulted in the formation of an instable neochromosome present in a single copy in the diploid cells, followed by its replacement by a shorter, partially palindromic neochromosome present in two copies, whose stability eventually increased the chromosome number of the diploid strains harboring it.

Impact of Covid-19 pandemic on arthroplasty services and early experience after resuming surgeries at a 'non Covid' center.

BACKGROUND: The aim of this study is to assess the impact of Covid-19 crisis on hip and knee joint replacement surgeries at a high volume tertiary care hospital in the Indian National Capital Region and to evaluate the early experience of resumption of arthroplasty services. METHODS: Institutional records of the arthroplasty cases, operated between 1st March to 31 August of 2019 (Group A, pre-Covid) and 2020 (Group B, pandemic year) were compared retrospectively over numerous parameters including the complications within six weeks of surgery. RESULTS: There was a significant drop (by 82.53 %) in the total number of arthroplasty surgeries in Group B (62) as compared with Group A (355). Average number of arthroplasties per month were 59.17 ± 12.93 and 10.67 ± 13.29 in Group A and Group B respectively (p < 0.001). There was a significant increase in postoperative complication rate 7/355 (1.97 %) in Group A vs 7/62 (11.29 %) in Group B during pandemic (p < 0.002), along with a higher 30-days mortality rate 2/355 (3.22 %) vs 2/62 (0.56 %). Pandemic year also saw an increased readmission rate (4.83 %) vs (0.56 %) and postoperative ICU transfer rate (1.61 %) vs (0.56 %) in comparison with pre-Covid year. CONCLUSION: In the pandemic, arthroplasty services got severely affected at our center. With nearly six fold increase in complication rates, higher 30-days mortality and increased readmission rates, caution is advised in resuming arthroplasty surgeries without robust evaluation of cases. Whether undetected Covid-19 infection or poor pre-existing disease control due to lockdown can be linked to these results is a matter of further research with larger multicenter studies.

Phenotypic and genomic hallmarks of a novel, potentially pathogenic rapidly growing Mycobacterium species related to the Mycobacterium fortuitum complex.

Previously, we have identified a putative novel rapidly growing Mycobacterium species, referred to as TNTM28, recovered from the sputum of an apparently immunocompetent young man with an underlying pulmonary disease. Here we provide a thorough characterization of TNTM28 genome sequence, which consists of one chromosome of 5,526,191 bp with a 67.3% G + C content, and a total of 5193 predicted coding sequences. Phylogenomic analyses revealed a deep-rooting relationship to the Mycobacterium fortuitum complex, thus suggesting a new taxonomic entity. TNTM28 was predicted to be a human pathogen with a probability of 0.804, reflecting the identification of several virulence factors, including export systems (Sec, Tat, and ESX), a nearly complete set of Mce proteins, toxin-antitoxins systems, and an extended range of other genes involved in intramacrophage replication and persistence (hspX, ahpC, sodA, sodC, katG, mgtC, ClpR, virS, etc.), some of which had likely been acquired through horizontal gene transfer. Such an arsenal of potential virulence factors, along with an almost intact ESX-1 locus, might have significantly contributed to TNTM28 pathogenicity, as witnessed by its ability to replicate efficiently in macrophages. Overall, the identification of this new species as a potential human pathogen will help to broaden our understanding of mycobacterial pathogenesis.

Impending spontaneous head neck dissociation caused by anteverted cup: A case report on reverse bottle opener effect and review of literature.

Modularity in total hip arthroplasty (THA) not only allows adjustments of leg length and offset but also simplifies the revision. It allows limited revision of various components and decreases surgical morbidity of complete revision. Despite benefits, modularity is associated with risks like corrosion and component dissociation. Dissociation between head and neck taper is rare and the cause is attributed to taper corrosion, revision, stem subsidence, pumping phenomenon, injury and closed reduction of dislocated THA. We report a case of late-onset impending head-neck dissociation in a THA caused by a well-fixed anteverted cup with polyethylene liner wear by "reverse bottle opener effect." To our knowledge, this is the most late-onset reported case of head-neck dissociation, occurring after 13yrs of index surgery.

Rare Asymptomatic Posterior Dislocation of Mobile Bearing Insert in Unicondylar Knee Arthroplasty: A Case Report.

Dislocation of polyethylene insert is a common complication of mobile bearing uniconylar knee arthroplasty (UKA). Dislocation, when occurs is common in anterior, lateral or medial direction. The authors report a case of posterior dislocation in which patient remained asymptomatic and the dislocation was detected in a routine follow-up radiograph done at 18 months after surgery. UKA was subsequently converted into total knee arthroplasty and patient had a stable knee at two years follow-up with no signs of loosening or instability. Posterior meniscal bearing dislocation may remain asymptomatic and continued examination with high index of suspicion is needed to disclose such complications in the absence of symptoms.

BBIQ, a pure TLR7 agonist, is an effective influenza vaccine adjuvant.

Better adjuvants are needed for vaccines against seasonal influenza. TLR7 agonists are potent activators of innate immune responses and thereby may be promising adjuvants. Among the imidazoquinoline compounds, 1-benzyl-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine (BBIQ) was reported to be a highly active TLR7 agonist but has remained relatively unexplored because of its commercial unavailability. Indeed, in silico molecular modeling studies predicted that BBIQ had a higher TLR7 docking score and binding free energy than imiquimod, the gold standard TLR7 agonist. To circumvent the availability issue, we developed an improved and higher yield method to synthesize BBIQ. Testing BBIQ on human and mouse TLR7 reporter cell lines confirmed it to be TLR7 specific with significantly higher potency than imiquimod. To test its adjuvant potential, BBIQ or imiquimod were admixed with recombinant influenza hemagglutinin protein and administered to mice as two intramuscular immunizations 2 weeks apart. Serum anti-influenza IgG responses assessed by ELISA 2 weeks after the second immunization confirmed that the mice that received vaccine admixed with BBIQ had significantly higher anti-influenza IgG1 and IgG2c responses than mice immunized with antigen alone or admixed with imiquimod. This confirmed BBIQ to be a TLR7-specific adjuvant able to enhance humoral immune responses.

TbD1 deletion as a driver of the evolutionary success of modern epidemic Mycobacterium tuberculosis lineages.

Mycobacterium tuberculosis (Mtb) strains are classified into different phylogenetic lineages (L), three of which (L2/L3/L4) emerged from a common progenitor after the loss of the MmpS6/MmpL6-encoding Mtb-specific deletion 1 region (TbD1). These TbD1-deleted "modern" lineages are responsible for globally-spread tuberculosis epidemics, whereas TbD1-intact "ancestral" lineages tend to be restricted to specific geographical areas, such as South India and South East Asia (L1) or East Africa (L7). By constructing and characterizing a panel of recombinant TbD1-knock-in and knock-out strains and comparison with clinical isolates, here we show that deletion of TbD1 confers to Mtb a significant increase in resistance to oxidative stress and hypoxia, which correlates with enhanced virulence in selected cellular, guinea pig and C3HeB/FeJ mouse infection models, the latter two mirroring in part the development of hypoxic granulomas in human disease progression. Our results suggest that loss of TbD1 at the origin of the L2/L3/L4 Mtb lineages was a key driver for their global epidemic spread and outstanding evolutionary success.

Prediction of novel mouse TLR9 agonists using a random forest approach.

BACKGROUND: Toll-like receptor 9 is a key innate immune receptor involved in detecting infectious diseases and cancer. TLR9 activates the innate immune system following the recognition of single-stranded DNA oligonucleotides (ODN) containing unmethylated cytosine-guanine (CpG) motifs. Due to the considerable number of rotatable bonds in ODNs, high-throughput in silico screening for potential TLR9 activity via traditional structure-based virtual screening approaches of CpG ODNs is challenging. In the current study, we present a machine learning based method for predicting novel mouse TLR9 (mTLR9) agonists based on features including count and position of motifs, the distance between the motifs and graphically derived features such as the radius of gyration and moment of Inertia. We employed an in-house experimentally validated dataset of 396 single-stranded synthetic ODNs, to compare the results of five machine learning algorithms. Since the dataset was highly imbalanced, we used an ensemble learning approach based on repeated random down-sampling. RESULTS: Using in-house experimental TLR9 activity data we found that random forest algorithm outperformed other algorithms for our dataset for TLR9 activity prediction. Therefore, we developed a cross-validated ensemble classifier of 20 random forest models. The average Matthews correlation coefficient and balanced accuracy of our ensemble classifier in test samples was 0.61 and 80.0%, respectively, with the maximum balanced accuracy and Matthews correlation coefficient of 87.0% and 0.75, respectively. We confirmed common sequence motifs including 'CC', 'GG','AG', 'CCCG' and 'CGGC' were overrepresented in mTLR9 agonists. Predictions on 6000 randomly generated ODNs were ranked and the top 100 ODNs were synthesized and experimentally tested for activity in a mTLR9 reporter cell assay, with 91 of the 100 selected ODNs showing high activity, confirming the accuracy of the model in predicting mTLR9 activity. CONCLUSION: We combined repeated random down-sampling with random forest to overcome the class imbalance problem and achieved promising results. Overall, we showed that the random forest algorithm outperformed other machine learning algorithms including support vector machines, shrinkage discriminant analysis, gradient boosting machine and neural networks. Due to its predictive performance and simplicity, the random forest technique is a useful method for prediction of mTLR9 ODN agonists.

A specialised SKI complex assists the cytoplasmic RNA exosome in the absence of direct association with ribosomes.

The Ski2-Ski3-Ski8 (SKI) complex assists the RNA exosome during the 3' to 5' degradation of cytoplasmic transcripts. Previous reports showed that the SKI complex is involved in the 3' to 5' degradation of mRNAs, including 3' untranslated regions (UTRs) and devoid of ribosomes. Paradoxically, we recently showed that the SKI complex directly interacts with ribosomes during the co-translational mRNA decay and that this interaction is necessary for its RNA degradation promoting activity. Here, we characterised a new SKI-associated factor, Ska1, that associates with a subpopulation of the SKI complex. We showed that Ska1 is specifically involved in the degradation of long 3'UTR-containing mRNAs, poorly translated mRNAs as well as other RNA regions not associated with ribosomes, such as cytoplasmic lncRNAs. We further show that the overexpression of SKA1 antagonises the SKI-ribosome association. We propose that the Ska1-SKI complex assists the cytoplasmic exosome in the absence of direct association of the SKI complex with ribosomes.

Diverse dynamics features of novel protein kinase C (PKC) isozymes determine the selectivity of a fluorinated balanol analogue for PKCε.

BACKGROUND: (-)-Balanol is an ATP-mimicking inhibitor that non-selectively targets protein kinase C (PKC) isozymes and cAMP-dependent protein kinase (PKA). While PKA constantly shows tumor promoting activities, PKC isozymes can ambiguously be tumor promoters or suppressors. In particular, PKCε is frequently implicated in tumorigenesis and a potential target for anticancer drugs. We recently reported that the C5(S)-fluorinated balanol analogue (balanoid 1c) had improved binding affinity and selectivity for PKCε but not to the other novel PKC isozymes, which share a highly similar ATP site. The underlying basis for this fluorine-based selectivity is not entirely comprehended and needs to be investigated further for the development of ATP mimic inhibitors specific for PKCε. RESULTS: Using molecular dynamics (MD) simulations assisted by homology modelling and sequence analysis, we have studied the fluorine-based selectivity in the highly similar ATP sites of novel PKC (nPKC) isozymes. The study suggests that every nPKC isozyme has different dynamics behaviour in both apo and 1c-bound forms. Interestingly, the apo form of PKCε, where 1c binds strongly, shows the highest degree of flexibility which dramatically decreases after binding 1c. CONCLUSIONS: For the first time to the best of our knowledge, we found that the origin of 1c selectivity for PKCε comes from the unique dynamics feature of each PKC isozyme. Fluorine conformational control in 1c can synergize with and lock down the dynamics of PKCε, which optimize binding interactions with the ATP site residues of the enzyme, particularly the invariant Lys437. This finding has implications for further rational design of balanol-based PKCε inhibitors for cancer drug development.

Short genome report of cellulose-producing commensal Escherichia coli 1094.

Bacterial surface colonization and biofilm formation often rely on the production of an extracellular polymeric matrix that mediates cell-cell and cell-surface contacts. In Escherichia coli and many Betaproteobacteria and Gammaproteobacteria cellulose is often the main component of the extracellular matrix. Here we report the complete genome sequence of the cellulose producing strain E. coli 1094 and compare it with five other closely related genomes within E. coli phylogenetic group A. We present a comparative analysis of the regions encoding genes responsible for cellulose biosynthesis and discuss the changes that could have led to the loss of this important adaptive advantage in several E. coli strains. Data deposition: The annotated genome sequence has been deposited at the European Nucleotide Archive under the accession number PRJEB21000.

A CRISPRi screen in E. coli reveals sequence-specific toxicity of dCas9.

High-throughput CRISPR-Cas9 screens have recently emerged as powerful tools to decipher gene functions and genetic interactions. Here we use a genome-wide library of guide RNAs to direct the catalytically dead Cas9 (dCas9) to block gene transcription in Escherichia coli. Using a machine-learning approach, we reveal that guide RNAs sharing specific 5-nucleotide seed sequences can produce strong fitness defects or even kill E. coli regardless of the other 15 nucleotides of guide sequence. This effect occurs at high dCas9 concentrations and can be alleviated by tuning the expression of dCas9 while maintaining strong on-target repression. Our results also highlight the fact that off-targets with as little as nine nucleotides of homology to the guide RNA can strongly block gene expression. Altogether this study provides important design rules to safely use dCas9 in E. coli.

The Gut Microbiota Facilitates Drifts in the Genetic Diversity and Infectivity of Bacterial Viruses.

The intestinal microbiota and human health are intimately linked, but interactions between bacteria and bacteriophages in the context of the mammalian intestine remain largely unexplored. We used comparative population genomics to study a tripartite network consisting of a virulent bacteriophage, its bacterial host, and a phage-insensitive bacterial strain both in vitro and within the murine gut. The bacteriophage adapted to infect the insensitive strain when the three partners co-existed in the gut of conventional mice, but not in dixenic mice or in planktonic cultures. The molecular changes associated with modifications in the bacteriophage host spectrum included single amino acid substitutions and an unusual homologous intragenomic recombination event within the genome of the bacteriophage. An intermediate bacterial host isolated from the murine microbiota mediated bacteriophage adaptation. Our data indicate that by offering access to new hosts, the microbiota shifts the genetic diversity of bacteriophages, thereby promoting long-term persistence of bacteriophage populations.

LIM-Only Protein FHL2 Is a Negative Regulator of Transforming Growth Factor ß1 Expression.

Transforming growth factor ß1 (TGF-ß1) is a master cytokine in many biological processes, including tissue homeostasis, epithelial-to-mesenchymal transition, and wound repair. Here, we report that four and a half LIM-only protein 2 (FHL2) is a critical regulator of TGF-ß1 expression. Devoid of a DNA-binding domain, FHL2 is a transcriptional cofactor that plays the role of coactivator or corepressor, depending on the cell and promoter contexts. We detected association of FHL2 with the TGF-ß1 promoter, which showed higher activity in Fhl2-/- cells than in wild-type (WT) cells in a reporter assay. Overexpression of FHL2 abrogates the activation of the TGF-ß1 promoter, whereas the upregulation of TGF-ß1 gene transcription correlates with reduced occupancy of FHL2 on the promoter. Moreover, ablation of FHL2 facilitates recruitment of RNA polymerase II on the TGF-ß1 promoter, suggesting that FHL2 may be involved in chromatin remodeling in the control of TGF-ß1 gene transcription. Enhanced expression of TGF-ß1 mRNA and cytokine was evidenced in the livers of Fhl2-/- mice. We tested the in vivo impact of Fhl2 loss on hepatic fibrogenesis that involves TGF-ß1 activation. Fhl2-/- mice developed more severe fibrosis than their WT counterparts. These results demonstrate the repressive function of FHL2 on TGF-ß1 expression and contribute to the understanding of the TGF-ß-mediated fibrogenic response.