Simplifying Recombinant Protein Production: Combining Golden Gate Cloning with a Standardized Protein Purification Scheme.

Recombinant protein production is pivotal in molecular biology, enabling profound insights into cellular processes through biophysical, biochemical, and structural analyses of the purified samples. The demand for substantial biomolecule quantities often presents challenges, particularly for eukaryotic proteins. Escherichia coli expression systems have evolved to address these issues, offering advanced features such as solubility tags, posttranslational modification capabilities, and modular plasmid libraries. Nevertheless, existing tools are often complex, which limits their accessibility and necessitate streamlined systems for rapid screening under standardized conditions. Based on the Golden Gate cloning method, we have developed a simple "one-pot" approach for the generation of expression constructs using strategically chosen protein purification tags like hexahistidine, SUMO, MBP, GST, and GB1 to enhance solubility and expression. The system allows visual candidate screening through mScarlet fluorescence and solubility tags are removable via TEV protease cleavage. We provide a comprehensive protocol encompassing oligonucleotide design, cloning, expression, His-tag affinity chromatography, and size-exclusion chromatography. This method, therefore, streamlines prokaryotic and eukaryotic protein production, rendering it accessible to standard molecular biology laboratories with basic protein biochemical equipment.

A single-chain fab derived drug conjugate for HER2 specific delivery.

Despite the development of antibody-drug conjugates, the fragment Fab-based drug conjugates offer some unique capabilities in terms of safety, clearance, penetration and others. Current methods for preparing Fab drug conjugates are limited by the availability and stability of Fab proteins, leaving reports on this rare. Here, we found that a single-chain scaffold of Fab enables stabilization of the paired structure and supports high-yield expression in bacteria cytoplasm. Furthermore, we conjugated anti-neoplastic agent SN38 to the C-terminus by sortase A ligation and generated a homogenous Fab conjugate with the drug-to-Fab ratio of 1. The resulting anti-HER2 Fab-SN38 conjugate demonstrated potent and antigen-dependent cell-killing ability with the aid of its special cathepsin-triggered cyclization-promoted release mechanism. In vivo, Fab-SN38 can prevent growths of HER2-positive tumors in athymic mice and be well tolerated to the treatment at 7 mg/kg per dose. Anti-tumor activity, high dose tolerance and penetration advantage observed in this study would merit Fab conjugate investigation in target chemotherapy.

His-Bundle Pacing for Pulmonary Hypertension With Bradycardia in Congenital Heart Disease: A Case Report.

Bradycardia, atrial tachyarrhythmia, heart failure, residual shunts, and pulmonary hypertension (PH) are significant problems after congenital heart disease surgery. We performed His-bundle pacing (HBP) for drug-resistant PH with bradycardia in a woman post-total anomalous pulmonary venous return repair complicated by bradycardia, hypertension, PH, residual atrial shunts, and heart failure. Significant improvement in PH post-HBP was achieved without exacerbation of left ventricular function. We successfully closed the atrial shunts 12 months after HBP.

Right ventriculography to guide left bundle branch pacing in pacing-induced cardiomyopathy: a novel case report.

Background: There is emerging evidence for the potential utility of left bundle branch area pacing (LBBAP), as an alternative to conventional cardiac resynchronization therapy (CRT). The utility of right ventriculography by way of power injector to facilitate lead placement has not yet been reported in the literature. Case summary: A 79-year-old female, with a background of poorly rate-controlled atrial fibrillation, presented with worsening dyspnoea. She had recently undergone single-chamber pacemaker insertion prior to an atrioventricular nodal (AVN) ablation, owing to failure in achieving successful CRT coronary sinus lead placement. She had clinical evidence of volume overload, and her electrocardiogram demonstrated right ventricular pacing. Echocardiography demonstrated left ventricular (LV) impairment, with an ejection fraction (EF) of 35%, and severe functional mitral regurgitation (MR). Her diagnosis was overall consistent with pacing-induced cardiomyopathy (PIC). In this patient, the use of right ventriculography, using power-injector-delivered contrast, successfully facilitated placement of an LBBAP lead, with confirmation of good threshold and sensing parameters. Following an upgrade to conduction system pacing, the patient recovered well. On recent follow-up, repeat echocardiography (24 months post initial presentation) demonstrated improved LV function (EF 45% from 35%) and only mild-to-moderate MR. Discussion: In conclusion, we demonstrate the utility of right ventriculography to facilitate placement of an LBBAP lead, successfully treating a patient who developed PIC from chronic right ventricular pacing following AVN ablation.

FCGR2/3 polymorphisms are associated with susceptibility to Kawasaki disease but do not predict intravenous immunoglobulin resistance and coronary artery aneurysms.

Introduction: Kawasaki disease (KD) is a pediatric vasculitis that can result in coronary artery aneurysm (CAA) formation, which is a dangerous complication. Treatment with intravenous immunoglobulin (IVIg) significantly decreases the risk of CAA, possibly through competitive binding to Fc-gamma receptors (FcγRs), which reduces the binding of pathological immune complexes. However, ~20% of children have recrudescence of fever and have an increased risk of CAA. Therefore, we aimed to identify genetic markers at the FCGR2/3 locus associated with susceptibility to KD, IVIg resistance, or CAA. Materials and methods: We investigated the association of single-nucleotide polymorphisms (SNPs) and copy number variations (CNVs) at the FCGR2/3 locus with KD susceptibility, IVIg resistance, and CAA risk using a family-based test (KD susceptibility) and case-control analyses (IVIg resistance and CAA risk) in different cohorts, adding up to a total of 1,167 KD cases. We performed a meta-analysis on IVIg resistance and CAA risk including all cohorts supplemented by previous studies identified through a systematic search. Results: FCGR2A-p.166His was confirmed to be strongly associated with KD susceptibility (Z = 3.17, p = 0.0015). In case-control analyses, all of the investigated genetic variations at the FCGR2/3 locus were generally not associated with IVIg resistance or with CAA risk, apart from a possible association in a Polish cohort for the FCGR3B-NA2 haplotype (OR = 2.15, 95% CI = 1.15-4.01, p = 0.02). Meta-analyses of all available cohorts revealed no significant associations of the FCGR2/3 locus with IVIg resistance or CAA risk. Discussion: FCGR2/3 polymorphisms are associated with susceptibility to KD but not with IVIg resistance and CAA formation. Currently known genetic variations at the FCGR2/3 locus are not useful in prediction models for IVIg resistance or CAA risk.

Cardiac resynchronization therapy guided by interventricular conduction delay: How to choose between biventricular pacing or conduction system pacing.

BACKGROUND: Biventricular pacing (BIV) is the gold standard for cardiac resynchronization therapy (CRT). Thirty percent of patients do not respond to CRT. Conduction system pacing (CSP) represents a viable alternative. Interventricular conduction delay (IVCD), as electrical desynchrony marker, is a CRT response predictor. The aim of this study was to determine the incidence of CRT responders by selecting the best approach between BIV and CPS based on intraoperative IVCD measurement in patients with HFrEF and LBBB. METHODS: Ninety-six patients were randomly assigned in a 1:1 ratio to either a standard BIV group(control group, CG) or a group where the CRT approach was determined based on IVCD evaluation(study group, SG). If the right ventricular sensed electrogram (RVs)-left ventricular sensed electrogram (LVs) interval was ≥100 ms, the lead was left in its original position; otherwise, the LV lead was removed, and CSP was performed instead. Clinical, EKG, and echocardiographic features have been assessed pre- and 6 months post-implant. Echocardiographic and clinical responder were evaluated. RESULTS: Thirty-seven percent of patients in the SG underwent CSP, as the operative algorithm. The incidence of CRT responders was significantly higher in the SG (echocardiographic criterion: 92.5% vs. 69.8%, p:.009; clinical criterion 87.5% vs. 62.8%, p:.014). The SG showed a significantly greater difference in EF between pre- and post-implant as well as reduced end-diastolic and systolic volumes. Univariate and multivariate regression analysis indicated that enrollment in the SG was the only factor associated with CRT response. CONCLUSION: Intraoperative assessment of IVCD could help determine the optimal CRT approach between BIV and CSP, leading to a significant improvement in the rate of CRT responders.

Stylet-driven Leads or Lumenless Leads for Conduction System Pacing.

Stylet-driven leads have been recently introduced for conduction system pacing, while most of the previous experience has been obtained with lumenless leads. Design and structural characteristics of both lead types are significantly different, resulting in different implant techniques and independent learning curves. Lead performance appears to be comparable, whereas data on direct comparison of clinical outcomes are scarce. Currently, there are no specific clinical scenarios favouring the use of one lead type over another and the decision should rely on the individual experience of the implanter.

Improving the diffraction quality of heat-shock protein 47 crystals.

Heat-shock protein 47 (HSP47) is a potential target for inhibitors that ameliorate fibrosis by reducing collagen assembly. In an effort to develop a structure-based drug-design system, it was not possible to replicate a previous literature result (PDB entry 4au4) for apo dog HSP47; instead, crystal forms were obtained in which pairs of dog HSP47 molecules interacted through a noncleavable C-terminal His-tag to build up tetramers, all of which had multiple molecules of HSP47 in the asymmetric unit and none of which diffracted as well as the literature precedent. To overcome these difficulties, a two-pronged approach was followed: (i) the His-tag was moved from the C-terminus to the N-terminus and was made cleavable, and (ii) Adnectin (derived from the tenth domain of human fibronectin type III) crystallization chaperones were developed. Both approaches provided well diffracting crystals, but the latter approach yielded crystal forms with only one or two HSP47 complexes per asymmetric unit, which made model building less onerous.

Fundoplication: Old Concept for Novel Challenges?

Background: Gastroesophageal reflux disease (GERD), the most common esophageal disorder worldwide, is a progressive condition that may lead to Barrett's esophagus and adenocarcinoma. Upfront therapy with proton pump inhibitors is ineffective in up to 40% of patients. The scope of surgical therapy is to reconstruct the natural antireflux barrier provided by the diaphragmatic crura, the lower esophageal sphincter, and the gastroesophageal flap valve. Summary: For 70 years, the 360° Nissen fundoplication has dominated the surgical scenario and is still considered the gold-standard treatment. However, over the past two decades, the Toupet and Dor partial fundoplications have emerged as alternative options to decrease the incidence of dysphagia and gas-bloat syndrome. Randomized and observational clinical studies have shown that the outcomes of partial fundoplication compare favorably with those of the Nissen and can provide satisfactory quality of life minimizing the risk of side effects. However, reflux control and anatomical integrity of partial fundoplications may fade away over time. Further research and close scrutiny of new surgical procedures and technologies is in progress to improve clinical outcomes and provide a more personalized and durable antireflux therapy. Key Messages: Laparoscopic antireflux surgery is a safe and effective therapy for GERD. It should be performed in centers offering a comprehensive diagnostic pathway and a spectrum of techniques tailored to the individual GERD phenotype.

Fundoplication is a surgical procedure to correct both hiatus hernia and gastroesophageal reflux disease. These conditions cause symptoms (acid reflux, heartburn, and regurgitation) and complications (chronic inflammation possibly leading to cancer). The fundoplication consists of encircling the distal part of the esophagus (food pipe) with a portion of the upper portion of the stomach to allow normal passage of food while preventing the return of food to the esophagus and the mouth. Over the past 70 years, the advantages and drawbacks of this operation have been widely reported. Still, the vast majority of patients and physicians have not fully embraced this therapeutic modality due to the fear of side effects such as difficult swallowing and bloating. Therefore, a partial fundoplication approach has been proposed to avoid these side effects and to provide a surgical repair that does not alter quality of life while maintaining the patient free from reflux and not dependent on drugs (proton pump inhibitors). However, partial fundoplications may not tighten enough the sphincter (valve) located between the esophagus and the stomach, and may not be durable over time. This paper describes the history of fundoplication, the mechanisms by which gastroesophageal reflux causes symptoms and complications, the indication for surgery, the main steps of laparoscopic (keyhole) surgery, and the search for innovative and minimally invasive techniques to strengthen the sphincter in order to cure symptoms and protect the esophagus from the deleterious effects of acid reflux.

Transvenous extraction of conduction system and lumenless pacing leads.

INTRODUCTION: Conduction system pacing (CSP), often accomplished with lumenless pacing leads, is increasingly employed to achieve physiologic ventricular activation. There are limited data on the extraction of these leads. The objective of this study was to describe the safety and efficacy of extraction of CSP pacing leads and compare outcomes with extraction of non-CSP lumenless leads. METHODS: Patients undergoing CSP/non-CSP lumenless lead removal were included. Outcomes of interest included rates of complete procedural success, complications, and successful reimplantation. RESULTS: Overall, 23 patients were included (n = 14 with CSP and n = 9 with non-CSP lumenless leads implanted in the right atrium, right ventricle, or septum). The mean age was 52.7 ± 24.0 years, 30% were female, and the mean lead age was 4.5 ± 4.4 years. The complete procedural success rate was 100%. One serious complication occurred in the non-CSP group but was unrelated to the lead of interest. Manual traction alone was successful in 57% of CSP cases (mean lead age 2.4 ± 1.7 years) and in 11% of non-CSP cases (mean lead age 7.9 ± 5.3 years). Laser sheaths were used in 43% of CSP cases and 89% of non-CSP cases; rotational cutting tools were used in no CSP cases and in 33% of non-CSP cases. Reimplantation in the conduction system was attempted with a left bundle branch pacing lead and successful in 80% (n = 4/5). CONCLUSION: Extraction of CSP and non-CSP lumenless leads is feasible with a high success rate and a good safety profile. CSP reimplantation after extraction is also feasible with good electrical performance.

A new dimension in cardiac imaging: Three-dimensional exploration of the atrioventricular conduction axis with hierarchical phase-contrast tomography.

Much of our understanding of the atrioventricular conduction axis has been derived from early 20th-century histologic investigations. These studies, although foundational, are constrained by their 2-dimensional representation of complex 3-dimensional anatomy. The variability in the course of the atrioventricular conduction axis, and its relationship to surrounding cardiac structures, necessitates a more advanced imaging approach. Using hierarchical phase-contrast tomography of an autopsied heart specimen with cellular resolution, this review provides a contemporary understanding of the atrioventricular conduction axis. By correlating these findings with 3-dimensional computed tomographic reconstructions in living patients, we offer clinicians the insights needed accurately to predict the location of the atrioventricular conduction axis. This novel approach overcomes the inherent limitations of 2-dimensional histology, enhancing our ability to understand and visualize the intricate relationships of the conduction axis within the heart.

Review of Atrioventricular Node Ablation Combined with Permanent His-Purkinje Conduction System Pacing in Patients with Atrial Fibrillation with Heart Failure.

With the advancement of pacing technologies, His-Purkinje conduction system pacing (HPCSP) has been increasingly recognized as superior to conventional right ventricular pacing (RVP) and biventricular pacing (BVP). This method is characterized by a series of strategies that either strengthen the native cardiac conduction system or fully preserve physical atrioventricular activation, ensuring optimal clinical outcomes. Treatment with HPCSP is divided into two pacing categories, His bundle pacing (HBP) and left bundle branch pacing (LBBP), and when combined with atrioventricular node ablation (AVNA), can significantly improve left ventricular (LV) function. It effectively prevents tachycardia and regulates ventricular rates, demonstrating its efficacy and safety across different QRS wave complex durations. Therefore, HPCSP combined with AVNA can alleviate symptoms and improve the quality of life in patients with persistent atrial fibrillation (AF) who are unresponsive to multiple radiofrequency ablation, particularly those with concomitant heart failure (HF) who are at risk of further deterioration. As a result, this "pace and ablate" strategy could become a first-line treatment for refractory AF. As a pacing modality, HBP faces challenges in achieving precise localization and tends to increase the pacing threshold. Thus, LBBP has emerged as a novel approach within HPCSP, offering lower thresholds, higher sensing amplitudes, and improved success rates, potentially making it a preferable alternative to HBP. Future large-scale, prospective, and randomized controlled studies are needed to evaluate patient selection and implantation technology, aiming to clarify the differential clinical outcomes between pacing modalities.

His Bundle Pacing and Left Bundle Branch Pacing in Patients with Heart Failure.

BACKGROUND: His bundle pacing (HBP) and left bundle branch pacing (LBBP) are emerging therapies for patients with heart failure and conduction disorders, offering potential advantages over traditional pacing methods. These approaches aim to restore physiological conduction and improve cardiac function more effectively. OBJECTIVE: This study aims to evaluate the efficacy and safety of HBP and LBBP in patients with heart failure and conduction disturbances, comparing these techniques to conventional pacing. METHODS: A comprehensive review of recent studies and clinical trials was conducted, focusing on the performance of HBP and LBBP in improving cardiac function, reducing QRS duration, and enhancing overall patient outcomes. The analysis includes data on clinical efficacy, procedural safety, and long-term benefits associated with these pacing modalities. RESULTS: Both HBP and LBBP have demonstrated significant improvements in cardiac function and clinical outcomes compared to conventional pacing. HBP effectively restores physiological conduction with improved synchronization and a reduction in QRS duration. LBBP has shown enhanced left ventricular activation, leading to better overall cardiac performance. Both techniques have been associated with a lower incidence of complications and a higher success rate in achieving optimal pacing thresholds. CONCLUSIONS: HBP and LBBP offer promising alternatives to traditional pacing for patients with heart failure and conduction disorders. These advanced pacing strategies provide superior clinical outcomes and improved cardiac function with reduced risk of complications. Further research and clinical trials are needed to fully establish the long-term benefits and safety profiles of these techniques in diverse patient populations.

Factors influencing health information system acceptance: a cross-sectional study from a low-middle-income country.

Background: The Avicenna unified Health Information System (HIS) was implemented by the Palestinian Ministry of Health in 2010 across government hospitals. Despite its potential, the acceptance of Avicenna HIS by healthcare providers remains unclear after 14 years of application. Understanding the factors that influence healthcare provider acceptance is essential for optimizing the system's success. We investigated factors affecting acceptance of Avicenna HIS among healthcare providers in Palestinian healthcare institutions, focusing on perceived usefulness, ease of use, human factors, technological factors, and organizational support. Materials and methods: A cross-sectional study was conducted at the Palestine Medical Complex (PMC) in Ramallah, West Bank, where the Avicenna HIS has been fully implemented since 2010. A systematic random sampling was used to select participants, resulting in 300 completed questionnaires. The study utilized a self-administered questionnaire adapted from a structured tool based on the Technology Acceptance Model (TAM). The questionnaire was validated through expert review and pilot testing, achieving a Cronbach's alpha of 0.86. Each selected healthcare provider was contacted face-to-face, and written informed consent was obtained before administering the questionnaire. Results: A total of 300 questionnaires were completed and returned. The study sample included 178 males (59.3%) and 122 females (40.7%). The majority of participants was aged 20-39 years (270 participants, 90%) and held a bachelor's degree (250 participants, 83.3%). Nurses comprised the largest professional group (153 participants, 51.0%). High levels of perceived usefulness and ease of use were reported, both with mean scores of 4.511 (S.D. = 0.295). Technological factors had a mean score of 4.004 (S.D. = 0.228), while organizational factors scored 2.858 (S.D. = 0.304). Overall acceptance of the HIS was moderately high, with a mean score of 4.218 (S.D. = 0.387). Significant differences in perceived usefulness and ease of use were noted based on gender, age, and experience. Conclusion: This study concludes that both technological and human factors significantly influence the acceptance of HIS among healthcare providers in Palestine. To improve HIS adoption, it is recommended to enhance system functionality, ensure reliable data quality, and provide comprehensive training programs for healthcare providers.

A Generic Transformation Approach for Complex Laboratory Data Using the Fast Healthcare Interoperability Resources Mapping Language: Method Development and Implementation.

Background: Reaching meaningful interoperability between proprietary health care systems is a ubiquitous task in medical informatics, where communication servers are traditionally used for referring and transforming data from the source to target systems. The Mirth Connect Server, an open-source communication server, offers, in addition to the exchange functionality, functions for simultaneous manipulation of data. The standard Fast Healthcare Interoperability Resources (FHIR) has recently become increasingly prevalent in national health care systems. FHIR specifies its own standardized mechanisms for transforming data structures using StructureMaps and the FHIR mapping language (FML). Objective: In this study, a generic approach is developed, which allows for the application of declarative mapping rules defined using FML in an exchangeable manner. A transformation engine is required to execute the mapping rules. Methods: FHIR natively defines resources to support the conversion of instance data, such as an FHIR StructureMap. This resource encodes all information required to transform data from a source system to a target system. In our approach, this information is defined in an implementation-independent manner using FML. Once the mapping has been defined, executable Mirth channels are automatically generated from the resources containing the mapping in JavaScript format. These channels can then be deployed to the Mirth Connect Server. Results: The resulting tool is called FML2Mirth, a Java-based transformer that derives Mirth channels from detailed declarative mapping rules based on the underlying StructureMaps. Implementation of the translate functionality is provided by the integration of a terminology server, and to achieve conformity with existing profiles, validation via the FHIR validator is built in. The system was evaluated for its practical use by transforming Labordatenträger version 2 (LDTv.2) laboratory results into Medical Information Object (Medizinisches Informationsobjekt) laboratory reports in accordance with the National Association of Statutory Health Insurance Physicians' specifications and into the HL7 (Health Level Seven) Europe Laboratory Report. The system could generate complex structures, but LDTv.2 lacks some information to fully comply with the specification. Conclusions: The tool for the auto-generation of Mirth channels was successfully presented. Our tests reveal the feasibility of using the complex structures of the mapping language in combination with a terminology server to transform instance data. Although the Mirth Server and the FHIR are well established in medical informatics, the combination offers space for more research, especially with regard to FML. Simultaneously, it can be stated that the mapping language still has implementation-related shortcomings that can be compensated by Mirth Connect as a base technology.

Advanced Deep Learning Fusion Model for Early Multi-Classification of Lung and Colon Cancer Using Histopathological Images.

BACKGROUND: In recent years, the healthcare field has experienced significant advancements. New diagnostic techniques, treatments, and insights into the causes of various diseases have emerged. Despite these progressions, cancer remains a major concern. It is a widespread illness affecting individuals of all ages and leads to one out of every six deaths. Lung and colon cancer alone account for nearly two million fatalities. Though it is rare for lung and colon cancers to co-occur, the spread of cancer cells between these two areas-known as metastasis-is notably high. Early detection of cancer greatly increases survival rates. Currently, histopathological image (HI) diagnosis and appropriate treatment are key methods for reducing cancer mortality and enhancing survival rates. Digital image processing (DIP) and deep learning (DL) algorithms can be employed to analyze the HIs of five different types of lung and colon tissues. METHODS: Therefore, this paper proposes a refined DL model that integrates feature fusion for the multi-classification of lung and colon cancers. The proposed model incorporates three DL architectures: ResNet-101V2, NASNetMobile, and EfficientNet-B0. Each model has limitations concerning variations in the shape and texture of input images. To address this, the proposed model utilizes a concatenate layer to merge the pre-trained individual feature vectors from ResNet-101V2, NASNetMobile, and EfficientNet-B0 into a single feature vector, which is then fine-tuned. As a result, the proposed DL model achieves high success in multi-classification by leveraging the strengths of all three models to enhance overall accuracy. This model aims to assist pathologists in the early detection of lung and colon cancer with reduced effort, time, and cost. The proposed DL model was evaluated using the LC25000 dataset, which contains colon and lung HIs. The dataset was pre-processed using resizing and normalization techniques. RESULTS: The model was tested and compared with recent DL models, achieving impressive results: 99.8% for precision, 99.8% for recall, 99.8% for F1-score, 99.96% for specificity, and 99.94% for accuracy. CONCLUSIONS: Thus, the proposed DL model demonstrates exceptional performance across all classification categories.

Facile Synthesis of Aptamer-Functionalized Polydopamine-Coated Magnetic Graphene Oxide Nanocomposites for Highly Efficient Purification of His-Tagged Proteins.

Recombinant proteins hold significant importance in numerous disciplines. As the demand for expressing and purifying these proteins grows, the scientific community is in dire need of a simple yet versatile methodology that can efficiently purify these proteins. Aptamers as synthetic nucleic acid-based ligands with high affinity have shown promise in this regard, as they can capture targets through molecular recognition. In this study, novel aptamer-functionalized polydopamine-coated magnetic graphene oxide nanocomposites were facilely prepared, achieving an impressive average aptamer coverage density (45 nmol/mg). These nanocomposites exhibited a uniform structure and robust magnetic responsiveness. The findings indicated that they possess several advantages, such as rapid adsorption, substantial capacity (171.4 mg/g), and excellent reusability. Notably, due to the inherent properties of nucleic acids, the immobilized aptamer-magnetic beads can be utilized repeatedly with high purification efficiency. Finally, the nanocomposites were further employed to purify His-tagged proteins from actual samples. Remarkably, they were able to selectively and efficiently isolate His-tagged retinoid X receptor alpha protein from complex Escherichia coli lysate. The purified His-tagged retinoid X receptor alpha protein was analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. This confirmed the efficacy of developed nanocomposites, reinforcing their vast potential for purification of His-tagged recombinant proteins.

Conduction system pacing compared with biventricular pacing for cardiac resynchronization therapy in patients with heart failure and mildly reduced left ventricular ejection fraction: Results from International Collaborative LBBAP Study (I-CLAS) Group.

BACKGROUND: Cardiac resynchronization therapy (CRT) is a guideline-recommended therapy in patients with heart failure with mildly reduced ejection fraction (HFmrEF, 36%-50%) and left bundle branch block or indication for ventricular pacing. Conduction system pacing (CSP) using left bundle branch area pacing or His bundle pacing has been shown to be a safe and physiologic alternative to biventricular pacing (BVP). OBJECTIVE: The aim of this study was to compare the clinical outcomes between BVP and CSP for patients with HFmrEF undergoing CRT. METHODS: Consecutive patients who underwent BVP or CSP with HFmrEF between January 2018 and June 2023 at 16 international centers were included. The primary outcome was the composite end point of time to death or heart failure hospitalization (HFH). Secondary end points included change in left ventricular ejection fraction (LVEF) and individual end points of death and HFH. RESULTS: A total of 1004 patients met inclusion criteria: BVP, 178; CSP, 826 (His bundle pacing, 154; left bundle branch area pacing, 672). Mean age was 73 ± 13 years; female, 34%; and LVEF, 42% ± 5%. Paced QRS duration in CSP was significantly narrower compared with BVP (129 ± 21 ms vs 144 ± 19 ms; P < .001). LVEF improved during follow-up in both groups (49% ± 10% vs 48% ± 10%; P = .32). CSP was independently associated with significant reduction in the primary end point of time to death or HFH compared with BVP (22% vs 34%; hazard ratio, 0.64; 95% confidence interval, 0.43-0.94; P = .025). CONCLUSION: CSP was associated with improved clinical outcomes compared with BVP in this large cohort of patients with HFmrEF undergoing CRT. Randomized controlled trials comparing CSP with BVP will be necessary to confirm these results.

Novel cellular functions of Cys2-His2 zinc finger proteins in anthracnose development and dissemination on pepper fruits by Colletotrichum scovillei.

Colletotrichum species are notorious for causing anthracnose on many fruits, leading to significant economic losses worldwide. As a model, we functionally characterized cys2-his2 (C2H2) zinc finger proteins (CsCZFs) in Colletotrichum scovillei, a major causal agent of pepper fruit anthracnose in many countries. In all, 62 CsCZFs were identified by in silico genomic analysis. Twelve were selected based on their expression profiles to generate targeted deletion mutants for functional investigation. ΔCsczf1 markedly reduced conidiation and constitutive expression of CsCZF1 partially recovered conidiation in an asexual reproduction-defective mutant, ΔCshox2. Deletion of CsCZF12, orthologous to the calcineurin-responsive transcription factor Crz1, impaired autophagy in C. scovillei. ΔCsczf9 was defective in surface recognition, appressorium formation, and suppression of host defenses. CsCZF9 was identified as an essential and novel regulator under the control of the mitogen-activated protein kinase (CsPMK1) in an early step of appressorium development in C. scovillei. This study provides novel insights into CsCZF-mediated regulation of differentiation and pathogenicity in C. scovillei, contributing to understanding the regulatory mechanisms governing fruit anthracnose epidemics.IMPORTANCEThe phytopathogenic fungus Colletotrichum scovillei is known to cause serious anthracnose on chili pepper. However, the molecular mechanism underlying anthracnose caused by this fungus remains largely unknown. Here, we systematically analyzed the functional roles of cys2-his2 zinc finger proteins (CsCZFs) in the dissemination and pathogenic development of this fungus. Our results showed that CsCZF1 plays an important role in conidiation and constitutive expression of CsCZF1 restored conidiation in an asexual reproduction-defective mutant, ΔCshox2. The CsCZF9, a novel target of the mitogen-activated protein kinase (CsPMK1), is essential for surface recognition to allow appressorium formation and suppression of host defenses in C. scovillei. The CsCZF12, orthologous to the calcineurin-responsive transcription factor Crz1, is involved in the autophagy of C. scovillei. Our findings reveal a comprehensive mechanism underlying CsCZF-mediated regulation of differentiation and pathogenicity of C. scovillei, which contributes to the understanding of fruit anthracnose epidemics and the development of novel strategies for disease management.

Evolution and development of the conduction system in the vertebrate heart: a role for hemodynamics and the epicardium.

Development of the heart is a very intricate and multiplex process as it involves not only the three spatial dimensions but also the fourth or time dimension. Over time, the heart of an embryo needs to adapt its function to serve the increasing complexity of differentiation and growth towards adulthood. It becomes even more perplexing by expanding time into millions of years, allocating related species in the tree of life. As the evolution of soft tissues can hardly be studied, we have to rely on comparative embryology, supported heavily by genetic and molecular approaches. These techniques provide insight into relationships, not only between species, but also between cell populations, signaling mechanisms, molecular interactions and physical factors such as hemodynamics. Heart development depends on differentiation of a mesodermal cell population that - in more derived taxa - continues in segmentation of the first and second heart field. These fields deliver not only the cardiomyocytes, forming the three-dimensionally looping cardiac tube as a basis for the chambered heart, but also the enveloping epicardium. The synchronized beating of the heart is then organized by the conduction system. In this Review, the epicardium is introduced as an important player in cardiac differentiation, including the conduction system.