Entry Site Research Articles

Determining the Main Infection Courts in Sweet Cherry Trees of the Canker Pathogens Calosphaeria pulchella, Cytospora sorbicola, and Eutypa lata.

The major fungal canker pathogens causing branch dieback of sweet cherry trees in California include Calosphaeria pulchella, Cytospora sorbicola, and Eutypa lata. These pathogens have long been known to infect cherry trees mainly through pruning wounds. However, recent field observations revealed numerous shoots and fruiting spurs exhibiting dieback symptoms with no apparent pruning wounds or mechanical injuries. Accordingly, this study was conducted to assess the incidence of the three pathogens in symptomatic terminal shoots and dying fruiting spurs, in addition to the wood below pruning wounds in branches. Surveys were conducted in five sweet cherry orchards across three counties in California. We also investigated the possibility that leaf scars, bud scars, and wounds resulting from fruit picking could serve as infection courts for Cal. pulchella, Cyt. sorbicola, and E. lata by means of artificial inoculations in the field. Orchard surveys revealed that Cal. pulchella had the highest pathogen incidence below pruning wounds in branch samples, followed by Cyt. sorbicola and E. lata. Among terminal shoots with dieback symptoms and dying fruiting spurs, Cyt. sorbicola was the most prevalent, followed by Cal. pulchella. Results from field inoculations indicated that fruit-picking wounds could serve as important infection courts for Cal. pulchella, Cyt. sorbicola, and E. lata, with average pathogen recovery of 41.5, 63, and 36.2%, respectively. Results also indicated that leaf and bud scars could serve as an entry site for Cyt. sorbicola, although recovery was relatively low. The present study is the first to identify harvest-induced wounds on fruiting spurs of sweet cherry as an important infection court of Cal. pulchella, Cyt. sorbicola, and E. lata.

HSP70 positively regulates translation by interacting with the IRES and stabilizes the viral structural proteins VP1 and VP3 to facilitate duck hepatitis A virus type 1 replication.

The maintenance of viral protein homeostasis depends on the interaction between host cell proteins and viral proteins. As a molecular chaperone, heat shock protein 70 (HSP70) has been shown to play an important role in viral infection. Our results showed that HSP70 can affect translation, replication, assembly, and release during the life cycle of duck hepatitis A virus type 1 (DHAV-1). We demonstrated that HSP70 can regulate viral translation by interacting with the DHAV-1 internal ribosome entry site (IRES). In addition, HSP70 interacts with the viral capsid proteins VP1 and VP3 and promotes their stability by inhibiting proteasomal degradation, thereby facilitating the assembly of DHAV-1 virions. This study demonstrates the specific role of HSP70 in regulating DHAV-1 replication, which are helpful for understanding the pathogenesis of DHAV-1 infection and provide additional information about the role of HSP70 in infection by different kinds of picornaviruses, as well as the interaction between picornaviruses and host cells.

Preliminary evidence of localizing CD8+ T-cell responses in COVID-19 patients with PET imaging.

The upper respiratory tract (URT) is the entry site for severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), from where it further disseminates. Early and effective adaptive immune responses are crucial to restrict viral replication and limit symptom development and transmission. Current vaccines increasingly incorporate strategies to boost mucosal immunity in the respiratory tract. Positron emission tomography (PET) is a non-invasive technology that measures cellular responses at a whole-body level. In this case series, we explored the feasibility of [89Zr]Zr-crefmirlimab berdoxam PET to assess CD8+ T-cell localization during active COVID-19. Our results suggest that CD8+ T-cell distributions assessed by PET imaging reflect their differentiation and functional state in blood. Therefore, PET imaging may represent a novel tool to visualize and quantify cellular immune responses during infections at a whole-body level.

Equine herpesvirus type 1 (EHV-1) replication at the upper respiratory entry site is inhibited by neutralizing EHV-1-specific IgG1 and IgG4/7 mucosal antibodies.

Equine herpesvirus type 1 (EHV-1) is a contagious respiratory pathogen that infects the mucosa of the upper respiratory tract (URT). Mucosal immune responses at the URT provide the first line of defense against EHV-1 and are crucial for orchestrating immunity. To define host-pathogen interactions, we characterized B-cell responses, antibody isotype functions, and EHV-1 replication of susceptible (non-immune) and clinically protected (immune) horses after experimental EHV-1 infection. Nasal secretion and nasal wash samples were collected and used for the isolation of DNA, RNA, and mucosal antibodies. Shedding of infectious virus, EHV-1 copy numbers, viral RNA expression, and host B-cell activation in the URT were compared based on host immune status. Mucosal EHV-1-specific antibody responses were associated with EHV-1 shedding and viral RNA transcription. Finally, mucosal immunoglobulin G (IgG) and IgA isotypes were purified and tested for neutralizing capabilities. IgG1 and IgG4/7 neutralized EHV-1, while IgG3/5, IgG6, and IgA did not. Immune horses secreted high amounts of mucosal EHV-1-specific IgG4/7 antibodies and quickly upregulated B-cell pathway genes, while EHV-1 was undetected by virus isolation and PCR. RNA transcription analysis reinforced incomplete viral replication in immune horses. In contrast, complete viral replication with high viral copy numbers and shedding of infectious viruses was characteristic for non-immune horses, together with low or absent EHV-1-specific neutralizing antibodies during viral replication. These data confirm that pre-existing mucosal IgG1 and IgG4/7 and rapid B-cell activation upon EHV-1 infection are essential for virus neutralization, regulation of viral replication, and mucosal immunity against EHV-1.IMPORTANCEEquine herpesvirus type 1 (EHV-1) causes respiratory disease, abortion storms, and neurologic outbreaks known as equine herpes myeloencephalopathy (EHM). EHV-1 is transmitted with respiratory secretions by nose-to-nose contact or via fomites. The virus initially infects the epithelium of the upper respiratory tract (URT). Host-pathogen interactions and mucosal immunity at the viral entry site provide the first line of defense against the EHV-1. Robust mucosal immunity can be essential in protecting against EHV-1 and to reduce EHM outbreaks. It has previously been shown that immune horses do not establish cell-associated viremia, the prerequisite for EHM. Here, we demonstrate how mucosal antibodies can prevent the replication of EHV-1 at the epithelium of the URT and, thereby, the progression of the virus to the peripheral blood. The findings improve the mechanistic understanding of mucosal immunity against EHV-1 and can support the development of enhanced diagnostic tools, vaccines against EHM, and the management of EHV-1 outbreaks.

Designing molecules: directing stem cell differentiation.

Stem cells have been widely applied in regenerative and therapeutic medicine for their unique regenerative properties. Although much research has shown their potential, it remains tricky in directing stem cell differentiation. The advancement of genetic and therapeutic technologies, however, has facilitated this issue through development of design molecules. These molecules are designed to overcome the drawbacks previously faced, such as unexpected differentiation outcomes and insufficient migration of endogenous or exogenous MSCs. Here, we introduced aptamer, bacteriophage, and biological vectors as design molecules and described their characteristics. The methods of designing/developing discussed include various Systematic Evolution of Ligands by Exponential Enrichment (SELEX) procedures, in silico approaches, and non-SELEX methods for aptamers, and genetic engineering methods such as homologous recombination, Bacteriophage Recombineering of Electroporated DNA (BRED), Bacteriophage Recombineering with Infectious Particles (BRIP), and genome rebooting for bacteriophage. For biological vectors, methods such as alternate splicing, multiple promoters, internal ribosomal entry site, CRISPR-Cas9 system and Cre recombinase mediated recombination were used to design viral vectors, while non-viral vectors like exosomes are generated through parental cell-based direct engineering. Besides that, we also discussed the pros and cons, and applications of each design molecule in directing stem cell differentiation to illustrate their great potential in stem cells research. Finally, we highlighted some safety and efficacy concerns to be considered for future studies.

Heterogeneous nuclear ribonucleoprotein A3 binds to the internal ribosomal entry site of enterovirus A71 and affects virus replication in neural cells.

Enterovirus A71 (EV-A71) belongs to the genus Enterovirus of the Picornaviridae family and often causes outbreaks in Asia. EV-A71 infection usually causes hand, foot, and mouth disease and can even affect the central nervous system, causing neurological complications or death. The 5'-untranslated region (5'-UTR) of EV-A71 contains an internal ribosome entry site (IRES) that is responsible for the translation of viral proteins. IRES-transacting factors can interact with the EV-A71 5'-UTR to regulate IRES activity. Heterogeneous nuclear ribonucleoprotein (hnRNP) A3 is a member of the hnRNP A/B protein family of RNA-binding proteins and is involved in RNA transport and modification. We found that hnRNP A3 knockdown promoted the replication of EV-A71 in neural calls. Conversely, increasing the expression of hnRNP A3 within cells inhibits the growth of EV-A71. HnRNP A3 can bind to the EV-A71 5'-UTR, and knockdown of hnRNP A3 enhances the luciferase activity of the EV-A71 5'-UTR IRES. The localization of hnRNP A3 shifts from the nucleus to the cytoplasm of infected cells during viral infection. Additionally, EV-A71 infection can increase the protein expression of hnRNP A3, and the protein level is correlated with efficient viral growth. Based on these findings, we concluded that hnRNP A3 plays a negative regulatory role in EV-A71 replication within neural cells.

Evaluation of the Effect of Morphological Structure on Dilatational Tracheostomy Interference Location and Complications with Ultrasonography and Fiberoptic Bronchoscopy.

Background: Percutaneous dilatational tracheostomy (PDT) is the most commonly performed minimally invasive intensive care unit procedure worldwide. Methods: This study evaluated the percentage of consistency between the entry site observed with fiberoptic bronchoscopy (FOB) and the prediction for the PDT level based on pre-procedural ultrasonography (USG) in PDT procedures performed using the forceps dilatation method. The effect of morphological features on intervention sites was also investigated. Complications that occurred during and after the procedure, as well as the duration, site, and quantity of the procedures, were recorded. Results: Data obtained from a total of 91 patients were analyzed. In 57 patients (62.6%), the USG-estimated tracheal puncture level was consistent with the intercartilaginous space observed by FOB, while in 34 patients (37.4%), there was a discrepancy between these two methods. According to Bland Altman, the agreement between the tracheal spaces determined by USG and FOB was close. Regression formulas for PDT procedures defining the intercartilaginous puncture level based on morphologic measurements of the patients were created. The most common complication related to PDT was cartilage fracture (17.6%), which was proven to be predicted with maximum relevance by punctured tracheal level, neck extension limitation, and procedure duration. Conclusions: In PDT procedures using the forceps dilatation method, the prediction of the PDT intervention level based on pre-procedural USG was considerably in accordance with the entry site observed by FOB. The intercartilaginous puncture level could be estimated based on morphological measurements.

Hyphal Als proteins act as CR3 ligands to promote immune responses against Candida albicans

Patients with decreased levels of CD18 (β2 integrins) suffer from life-threatening bacterial and fungal infections. CD11b, the α subunit of integrin CR3 (CD11b/CD18, αMβ2), is essential for mice to fight against systemic Candida albicans infections. Live elongating C. albicans activates CR3 in immune cells. However, the hyphal ligands that activate CR3 are not well defined. Here, we discovered that the C. albicans Als family proteins are recognized by the I domain of CD11b in macrophages. This recognition synergizes with the β-glucan-bound lectin-like domain to activate CR3, thereby promoting Syk signaling and inflammasome activation. Dectin-2 activation serves as the “outside-in signaling” for CR3 activation at the entry site of incompletely sealed phagosomes, where a thick cuff of F-actin forms to strengthen the local interaction. In vitro, CD18 partially contributes to IL-1β release from dendritic cells induced by purified hyphal Als3. In vivo, Als3 is vital for C. albicans clearance in mouse kidneys. These findings uncover a novel family of ligands for the CR3 I domain that promotes fungal clearance.

PSPC1 Binds to HCV IRES and Prevents Ribosomal Protein S5 Binding, Inhibiting Viral RNA Translation.

Hepatitis C virus (HCV) infects the human liver, and its chronic infection is one of the major causes of Hepatocellular carcinoma. Translation of HCV RNA is mediated by an Internal Ribosome Entry Site (IRES) element located in the 5'UTR of viral RNA. Several RNA Binding proteins of the host interact with the HCV IRES and modulate its function. Here, we demonstrate that PSPC1 (Paraspeckle Component 1), an essential paraspeckle component, upon HCV infection is relocalized and interacts with HCV IRES to prevent viral RNA translation. Competition UV-crosslinking experiments showed that PSPC1 interacts explicitly with the SLIV region of the HCV IRES, which is known to play a vital role in ribosomal loading to the HCV IRES via interaction with Ribosomal protein S5 (RPS5). Partial silencing of PSPC1 increased viral RNA translation and, consequently, HCV replication, suggesting a negative regulation by PSPC1. Interestingly, the silencing of PSPC1 protein leads to an increased interaction of RPS5 at the SLIV region, leading to an overall increase in the viral RNA in polysomes. Overall, our results showed how the host counters viral infection by relocalizing nuclear protein to the cytoplasm as a survival strategy.

Effect of cranial entry site on the rate of proximal catheter misplacement in ventriculoperitoneal shunt insertion.

The insertion of a ventriculoperitoneal shunt (VPS) is a common neurosurgical procedure, but the optimal entry site of the ventricular catheter is still under debate. In this study, the authors compare the parietal (Keen's) and frontal (Kocher's) entry sites in terms of the rate of revision surgery due to ventricular catheter misplacement, VPS dysfunction, and VPS infection. The authors retrospectively analyzed the data on consecutive adults (age ≥ 18 years) who had undergone primary VPS insertion between 2010 and 2020 at two neurosurgical centers. One center regularly inserts the ventricular catheter frontally (frontal group); the other center, parietally (parietal group). The primary outcome of interest was the rate of ventricular catheter misplacement necessitating revision surgery. Secondary outcomes were functional outcome as measured by the modified Rankin Scale (mRS), rate of revision surgery for VPS dysfunction and infection, as well as early (≤ 30 days) and late (> 30 days) mortality rates. Propensity score matching was performed based on baseline variables, such as normal pressure hydrocephalus, postinfectious hydrocephalus, and idiopathic intracranial hypertension, which were identified as predictors of ventricular catheter misplacement using logistic regression analysis. Among 539 consecutive patients, 301 (55.8%) were in the frontal group and 238 (44.2%) in the parietal group. Postoperative rates of revision surgery due to misplacement were comparable in the two catheter entry site groups (frontal 14 [4.7%] vs parietal 11 [4.6%], p = 0.987). Rates of revision surgery for VPS dysfunction (14 [4.7%] vs 10 [4.2%], respectively, p = 0.802) and infection (22 [7.3%] vs 10 [4.2%], p = 0.13) exhibited no significant differences. Favorable functional outcomes (mRS score ≤ 2; 164 [76.3%] vs 174 [79.5%], respectively, p = 0.058) and early mortality rates (5 [1.7%] vs 6 [2.5%], p = 0.483) were similar between the groups. After propensity score matching, the primary and secondary outcome measures remained comparable between the groups. The entry site of the ventricular catheter in VPS surgery does not seem to affect proximal revision rates. Further, revision rates due to VPS dysfunction, VPS infection, and morbidity were comparable as well.

An Outcome Analysis of Pediatric Diaphyseal Fractures Treated Surgically With the Titanium Elastic Nailing System.

Introduction Pediatric fractures account for one-fourthof all pediatric injuries. Stabilizing the fracture, regulating the length and alignment, encouraging bone healing, and minimizing morbidity and problems for the child and family are the objectives of treatment for diaphyseal fractures of long bones in children. Our goal is to investigate how pediatric diaphyseal long bone fractures are treated with a titanium elastic nailing system (TENS). Methods A prospective interventional study was conducted on 24 children who had displaced diaphyseal fractures of major long bones, involving 31 diaphyseal fractures of long bones. Utilizing Flynn's grading standards, the result was examined. Results The mean age was 12.20 years. The youngest child was seven years old and the eldest child was 16 years old. There were 20 boys (83.33%) and four girls (16.67%). The male-to-female ratio was noted to be 5:1. The commonest mode of injury was road traffic accidents (12 cases, 50%), followed by falls while playing (10 cases, 41.67%). Other causes included falls from height (one case, 4.17%)and blunt trauma (one case, 4.17%). The commonest bone to get fractured was the femur (37.50%), followed by both bones of the forearm (29.17%), tibia (20.83%), humerus (8.33%), and ulna alone (4.17%). The middle third (21 fractures, 67.74%) was the most prevalent location for fractures. Five fractures each (16.13%) accounted for in the proximal and distal thirds. Twelve fractures (38.71%) were detected on the left side, while the majority of fractures (19 fractures, 61.29%) were seen on the right side. Most of the fractures in this group were transverse fractures (18 fractures, 58.06%) followed by oblique fractures (eightfractures, 25.81%). Comminuted fractures accounted for five fractures (16.13%). Of the 31 fractures, open reduction had to be done in two fractures, after unsuccessful attempts at closed reduction. Closed reduction was done in 29 fractures. There were 15.12 weeks in the average union term. The range is six weeks to 39 weeks. The most frequent side effect was discovered to be skin irritation at the entry site. The extraosseous portion of nails caused irritation at two entry sites (6.45%). A case had delayed union (3.23%) and restricted knee range of movements. Conclusion For the treatment of juvenile diaphyseal fractures of the long bones, the TENSis the best option. It is a quick, straightforward, safe, dependable, and efficient way to treat pediatric long-bone fractures in patients aged five to 16 years. The healing process takes a fair amount of time, while the surgery takes less time. It does away with the necessity for extended bed rest and significantly shortens hospital stays. It provides stability and elastic mobility, which is perfect for early mobilization and quick union at the fracture site. It has a low rate of complications and produces excellent functional results.

Left bundle branch area pacing improves right ventricular function and synchrony

BackgroundThe impact of left bundle branch area pacing (LBBAP) on right ventricular (RV) function and tricuspid regurgitation (TR) remains unclear. ObjectiveWe aimed to assess the long-term effects of LBBAP on RV performance and on TR. MethodsRV function was evaluated using RV free wall strain, tricuspid annular plane systolic excursion, fractional area changing, and systolic velocity of the lateral tricuspid annulus. The presence of reverse septal flash (RSF) and basal bulge (BB) was used to assess RV motion pattern. The distance between the lead entry site on the interventricular septum and the septal leaflet of the tricuspid annulus (lead-TV distance) was measured. ResultsThe analysis included 122 subjects [62 men (50.8%); mean age 76.5 ± 11.4 years] with a median follow-up of 21 months (18–24.5 months). During follow-up, RV free wall strain improved significantly (15.2 ± 5.8 vs 16.4 ± 5.5; P < .001) while tricuspid annular plane systolic excursion, systolic, and fractional area changing remained unchanged. Left ventricular ejection fraction was an independent predictor of improved RV function (B = 3.51; 95% confidence interval 1.39–8.9; P = .01). With LBBAP, RSF disappeared in 22 of 23 patients (96%) and BB in 15 of 22 patients (68%) in whom RSF and BB were present at baseline, respectively. RV function improvement was significantly higher when RSF was present at baseline (14 patients vs 11 patients; P = .02). At follow-up, no significant deterioration in TR occurred for the overall group. However, a lead-TV distance of <24.5 mm was associated with TR progression. ConclusionLBBAP has a favorable impact on RV function. A basal LBBAP position is associated with worsening TR.

Fluorescent-protein co-expression to select CHO cells expressing high quantities of vaccine antigens.

Cell line development for production of vaccine antigens or therapeutic proteins typically involves transfection, selection, and enrichment for high-expressing cells. Enrichment methods include minipool enrichment, antibody-based enrichment, and enrichment based on co-expressed fluorescent biosensor proteins. However, these methods have limitations regarding labor and cost intensity, the generation of antibodies and assurance of their viral safety, and potential expression-interference or signal-saturation of the co-expressed fluorescent protein. To improve the method of fluorescent-protein co-expression, expression constructs were created that constitutively express a model vaccine antigen together with one of three fluorescent proteins having translation initiation controlled by a wildtype or mutant internal ribosome entry site (IRES), for a total of six constructs. The constructs were transfected into Chinese hamster ovary cells (CHO) cells, enriched for high fluorescence, cultured, and tested in a mini bioreactor to identify the most promising construct. The fluorescent protein, Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) with a mutant IRES performed best and was further tested with three additional vaccine antigens. Across the four vaccine antigens, the FUCCI fluorescent protein yielded productivity enhancements, without the need for generating an antibody and assuring its viral safety. Furthermore, FUCCI protein was present in negligible quantities in the cell supernatant, indicating a low risk for contaminating drug substances or vaccine antigen.

Molecular characterization of coxsackievirus A24 variants isolated from an outbreak of acute hemorrhagic conjunctivitis

PurposeAcute Hemorrhagic conjunctivitis (AHC) is associated with CVA24v. Recently there was a severe outbreak of conjunctivitis in months of July and August 2023 in India. This study emphasizes the identification of the distinct mutations in the CVA24v strains, which were isolated during the AHC outbreak and could have potentially played a role in the high transmission of AHC in India during the 2023 outbreak. MethodsA total of 71 conjunctivitis patients aged 1–75 years comprising 47 males and 24 females who attended Ophthalmology department of a tertiary care hospital of easternIndia were studied.RNA was extracted from all conjunctival swab samples and converted into cDNA. Subsequently, the viral 5′ UTR was amplified and the PCR positive samples were subjected to sequencing. The newly isolated viral 5′ UTR sequences were aligned with other worldwide sequences using the Clustal W tool to conduct mutational analysis. A phylogenetic tree was built using the MEGA software for viral genotype identification. ResultsAll of the current outbreak strains belonged to genotype IV of CVA24v. The present outbreak strains formed a distinct clade in the phylogenetic tree and were different from previously reported Indian strains. Two persistent mutations, specifically in domain IV (T213C) and domain V (C475T), were exclusively detected within the internal ribosome entry site (IRES) of the 5′ UTR of the current strains causing the outbreak. These two alterations have previously been shown to impact the virulence of another enterovirus (CV B3), but they have not been described in CVA24v until now. ConclusionFinding of the present study highlights the possibility and the significance of the aforementioned two mutations in enhancing the transmissibility of the newer CVA24v strains. Hence, these two distinct mutations should be investigated further for developing antiviral therapies to combat future AHC outbreaks associated with CVA24v.

The C-terminus of the prototypical M2 muscarinic receptor localizes to the mitochondria and regulates cell respiration under stress conditions.

Muscarinic acetylcholine receptors are prototypical G protein-coupled receptors (GPCRs), members of a large family of 7 transmembrane receptors mediating a wide variety of extracellular signals. We show here, in cultured cells and in a murine model, that the carboxyl terminal fragment of the muscarinic M2 receptor, comprising the transmembrane regions 6 and 7 (M2tail), is expressed by virtue of an internal ribosome entry site localized in the third intracellular loop. Single-cell imaging and import in isolated yeast mitochondria reveals that M2tail, whose expression is up-regulated in cells undergoing integrated stress response, does not follow the normal route to the plasma membrane, but is almost exclusively sorted to the mitochondria inner membrane: here, it controls oxygen consumption, cell proliferation, and the formation of reactive oxygen species (ROS) by reducing oxidative phosphorylation. Crispr/Cas9 editing of the key methionine where cap-independent translation begins in human-induced pluripotent stem cells (hiPSCs), reveals the physiological role of this process in influencing cell proliferation and oxygen consumption at the endogenous level. The expression of the C-terminal domain of a GPCR, capable of regulating mitochondrial function, constitutes a hitherto unknown mechanism notably unrelated to its canonical signaling function as a GPCR at the plasma membrane. This work thus highlights a potential novel mechanism that cells may use for controlling their metabolism under variable environmental conditions, notably as a negative regulator of cell respiration.

The Odds of Protein Translation Control Under Stress.

Physical or chemical stress is commonly known to inhibit protein translation at the cellular level. Since the process of protein translation requires catalysis by a multi-component machinery containing eukaryotic initiation factors (eIFs) and ribosomes in a sequence of reactions, how the process fails to proceed and whether certain genes can escape such blockade have provoked research efforts. Lines of evidence have demonstrated that phosphorylation of eIF4E or dephosphorylation of 4E-binding proteins (4E-BPs) prevents the formation of the eukaryotic translation initiation factor 4F (eIF4F) complex, whereas phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α) due to activation of heme-regulated inhibitor (HRI), general control nonderepressible 2 (GCN2), protein kinase RNA-like endoplasmic reticulum kinase (PERK), or protein kinase R (PKR) by a diverse array of stressors prevents eIF2-GTP-tRNAiMet ternary complex assembly. These signal the abandonment of translation initiation via 5'-7-methylguanine (m7G) cap recognition by eIF4E. Stress can promote cleavage of tRNAs, impediment of rRNA processing, changes in the epitranscriptomic landscape, ribosome stalling or collision, activation of ribosomal surveillance systems, and assembly of the stress granules. Although these events contribute to the general inhibition of protein translation, a few proteins can bypass such negativity and become translated selectively. Such selective protein translation is primarily m7G cap independent through the integrated stress response or Internal Ribosomal Entry Site (IRES). The newly synthesized proteins often influence cell fate, facilitate cell survival, and build endogenous defense. Insights into the general inhibition of protein translation and selective translation of specific proteins will advance our understanding of the etiology or progression of human diseases involving cellular stress from viral infection or inflammation to myocardial infarction, stroke, or neurodegenerative disease. Antioxid. Redox Signal. 40, 943-947.

Genetic mechanisms underlying the structural elaboration and dissemination of viral internal ribosomal entry sites.

Viral internal ribosomal entry sites (IRESs) form several classes that use distinct mechanisms to mediate end-independent initiation of translation. The origin of viral IRESs is a longstanding question. The simplest IRESs comprise tandem pseudoknots and occur in the intergenic region (IGR) of Dicistroviridae genomes (order Picornavirales ). Larger IGR IRESs contain additional elements that determine specific properties such as binding to the head of the ribosoma l 40S subunit. Metagenomic analyses reported here identified novel groups of structurally distinct IGR-like IRESs. The smallest of these (∼120nt long) comprise three pseudoknots and bind directly to the ribosomal P site. Others are up to 260nt long: insertions occurred at specific loci, possibly reflecting non-templated nucleotide insertion during replication. Various groups can be arranged in order, differing by the cumulative addition of single structural elements, suggesting an accretion mechanism for the structural elaboration of IRESs. Identification of chimeric IRESs implicates recombinational exchange of domains as a second mechanism for the diversification of IRES structure. Recombination likely also accounts for the presence of IGR-like IRESs at the 5'-end of some dicistrovirus-like genomes (e.g. Hangzhou dicistrovirus 3) and in the RNA genomes of Tombusviridae (order Tolivirales ), Marnaviridae (order Picornavirale s), and the 'Ripiresk' picorna-like clade (order Picornavirale s).

Substitutions of stem-loop subdomains in internal ribosome entry site of Senecavirus A: Impacts on rescue of sequence-modifying viruses

Senecavirus A (SVA) has a positive-sense, single-stranded RNA genome. Its 5´ untranslated region harbors an internal ribosome entry site (IRES), comprising 10 larger or smaller stem-loop structures (including a pseudoknot) that have been demonstrated to be well conserved. However, it is still unclear whether each stem-loop subdomain, such as a single stem or loop, is also highly conserved. To clarify this issue in the present study, a set of 29 SVA cDNA clones were constructed by site-directed mutagenesis (SDM) on the IRES. The SDM-modified scenarios included: (1) stem-formed complementary sequences exchanging with each other; (2) loop transversion; (3) loop transition; and (4) point mutations. All cDNA clones were separately transfected into cells for rescuing viable viruses, whereas only four SVAs of interest could be recovered, and were genetically stable during 20 passages. One progeny grew significantly slower than the other three did. The dual-luciferase reporter assay showed that none of the SDM-modified IRESes significantly inhibited the IRES activity. Our previous study indicated that a single motif from any of the ten stem structures, if completely mutated, would cause the failure of virus recovery. Interestingly, our present study revealed three stem structures, whose individual complementary sequences could exchange with each other to rescue sequence-modifying SVAs. Moreover, one apical loop was demonstrated to have the ability to tolerate its own full-length transition, also having no impact on the recovery of sequence-modifying SVA. The present study suggested that not every stem-loop structure was strictly conserved in its conformation, while the full-length IRES itself was well conserved. This provides a new research direction on interaction between the IRES and many factors.

The mesenteric entry site as a potential weak point in gastrointestinal anastomoses – findings from an ex-vivo biomechanical analysis

PurposeGastrointestinal disorders frequently necessitate surgery involving intestinal resection and anastomosis formation, potentially leading to severe complications like anastomotic leakage (AL) which is associated with increased morbidity, mortality, and adverse oncologic outcomes. While extensive research has explored the biology of anastomotic healing, there is limited understanding of the biomechanical properties of gastrointestinal anastomoses, which was aimed to be unraveled in this study.MethodsAn ex-vivo model was developed for the biomechanical analysis of 32 handsewn porcine end-to-end anastomoses, using interrupted and continuous suture techniques subjected to different flow models. While multiple cameras captured different angles of the anastomosis, comprehensive data recording of pressure, time, and temperature was performed simultaneously. Special focus was laid on monitoring time, location and pressure of anastomotic leakage (LP) and bursting pressures (BP) depending on suture techniques and flow models.ResultsSignificant differences in LP, BP, and time intervals were observed based on the flow model but not on the suture techniques applied. Interestingly, anastomoses at the insertion site of the mesentery exhibited significantly higher rates of leakage and bursting compared to other sections of the anastomosis.ConclusionThe developed ex-vivo model facilitated comparable, reproducible, and user-independent biomechanical analyses. Assessing biomechanical properties of anastomoses offers an advantage in identifying technical weak points to refine surgical techniques, potentially reducing complications like AL. The results indicate that mesenteric insertion serves as a potential weak spot for AL, warranting further investigations and refinements in surgical techniques to optimize outcomes in this critical area of anastomotic procedures.

A 5' UTR Language Model for Decoding Untranslated Regions of mRNA and Function Predictions.

The 5' UTR, a regulatory region at the beginning of an mRNA molecule, plays a crucial role in regulating the translation process and impacts the protein expression level. Language models have showcased their effectiveness in decoding the functions of protein and genome sequences. Here, we introduced a language model for 5' UTR, which we refer to as the UTR-LM. The UTR-LM is pre-trained on endogenous 5' UTRs from multiple species and is further augmented with supervised information including secondary structure and minimum free energy. We fine-tuned the UTR-LM in a variety of downstream tasks. The model outperformed the best known benchmark by up to 5% for predicting the Mean Ribosome Loading, and by up to 8% for predicting the Translation Efficiency and the mRNA Expression Level. The model also applies to identifying unannotated Internal Ribosome Entry Sites within the untranslated region and improves the AUPR from 0.37 to 0.52 compared to the best baseline. Further, we designed a library of 211 novel 5' UTRs with high predicted values of translation efficiency and evaluated them via a wet-lab assay. Experiment results confirmed that our top designs achieved a 32.5% increase in protein production level relative to well-established 5' UTR optimized for therapeutics.