Gastric cancer (GC) development follows a sequential and complex pathophysiological process known as Correa's cascade, progressing from chronic gastritis (active or inactive) to precancerous lesions (intestinal metaplasia (IM) and dysplasia) and ultimately to adenocarcinoma. Current GC therapies have limited efficacy and high recurrence rates. Autophagy, a cellular self-degradation mechanism that removes damaged or harmful components, plays a critical role in maintaining cellular homeostasis. Phytochemicals, owing to their low toxicity and multi-target pharmacological properties, exhibit anti-inflammatory, antimicrobial, antioxidant, and anticancer activities, and certain phytochemicals have shown potential in suppressing GC progression. In the inflammatory stage, metabolites such as berberine (an alkaloid) and chlorogenic acid (a phenolic acid) modulate autophagy by regulating the phosphoinositide 3-kinase (PI3K)/AKT/mTOR pathway: upstream activation of PI3K leads to AKT phosphorylation, which subsequently inhibits the downstream autophagy-related genes Unc-51-like kinase (ULK1) and Beclin-1, while these phytochemicals can reverse this inhibition to reduce inflammatory cytokine secretion and promote ulcer healing. In the precancerous stage, agents such as ginsenoside Rg3 and astragaloside IV (terpenoids) regulate autophagy through the PI3K/AKT axis and by influencing p62/sequestosome 1 (SQSTM1), an autophagy adaptor that links upstream microtubule-associated protein 1 light chain 3 (LC3)-interacting partners to downstream ubiquitinated substrates-thereby restoring autophagic flux and preventing malignant transformation. In the cancer stage, phytochemicals from various subclasses (phenolics, alkaloids, and terpenoids) target the core autophagy machinery: they suppress upstream PI3K/AKT/mTOR signaling (with mTOR directly inhibiting the initiation complex formed by ULK1, ATG13, and FIP200), induce the expression of Beclin-1 and ATG family proteins, promote the conversion of LC3-I to LC3-II, and enhance autophagosome formation. Consequently, these metabolites inhibit GC cell proliferation, migration, and angiogenesis while inducing apoptosis and increasing chemosensitivity. From a clinical translation perspective, the consistent observation that pharmacological inhibition of cytoprotective autophagy enhances phytochemical-induced apoptosis suggests a rational combination strategy: pairing a phytochemical that induces cytoprotective autophagy with an approved autophagy inhibitor such as hydroxychloroquine. This approach has been tested in vitro for curcumin, quercetin, and berberine but not yet in animal models or clinical trials. Given the established safety profiles of these agents individually, such combinations could be rapidly tested in preclinical gastric cancer models and, if successful, progressed to early-phase clinical trials as adjuncts to standard chemotherapy. Moreover, ongoing clinical trials of berberine for gastrointestinal metaplasia and of resveratrol for chemotherapy toxicity reduction provide existing clinical frameworks onto which autophagy endpoint measurements could be incorporated. These findings highlight the potential of autophagy-targeting phytochemicals as promising strategies for GC prevention and treatment, offering valuable insights for clinical translation. Moreover, targeting autophagy with phytochemicals represents a promising avenue for future drug development, potentially enabling personalized combination therapies to overcome chemoresistance and improve long-term outcomes for patients with gastric cancer.

The CRISPR-Cas9 system has revolutionized genome editing, but generating multiple gene knockouts (multi-KOs) remains challenging. This study presents an improved method for efficient generation of multi-KOs using sgRNA DNA strands and the CRISPR/Cas9 system. The method was tested on genes involved in the apoptosis pathway of HEK293T cells. The crRNA for each target gene was designed from Exon 1, near the starting codon ATG, such that a small deletion or insertion in this region would lead to a premature STOP codon and functional knockout. The T7 endonuclease I assay confirmed the high editing efficiency of sgRNAs in both single- and multiple-gene knockouts. Deep sequencing analysis revealed frameshift mutations and premature stop codons, indicating successful loss-of-function mutations. Using the sgRNA DNA strands provided a more uniform and efficient editing profile compared to other methods. This improved protocol offers a simpler, faster, and cost-effective approach for generating multigene knockout clones, with implications for functional genomics, therapeutic development, and synthetic biology.

Methyl-CpG-binding domain protein 4 (MBD4) is a base excision repair glycosylase that removes the thymine of a T:G mispair. If left unrepaired, these mismatches can cause C>T transitions. Human MBD4 uses an arginine finger, R468, to flip the mispaired thymine into its active site and to stabilize the orphaned guanine. MBD4 also possesses a second highly conserved arginine, R512, in close proximity to R468, whose role remains unknown despite an R512W mutation catalogued in colorectal cancers and an R512Q mutation reported in Myelodysplastic Syndromes. Here, we investigate the structural and functional significance of R512 using MBD4 variants R512A, R512K, R512Q, and R512W. We find that the native hydrogen-bonding network mediated by R512 is critical for active-site stability, and that R512K is the only variant to retain catalytic activity. This mutant preserves the wild-type rate of base excision despite a reduced affinity for the DNA substrate. Structural characterization of R512K MBD4 bound to T:G-mismatched DNA reveals an ordered water molecule in this pocket. This water molecule, along with the lysine amino group, retain 6 of the 7 native R512 hydrogen bonds, thereby preserving the local architecture of the enzyme. Comparative structural modeling of the catalytically inactive R512A, R512Q, and R512W variants suggests that loss of localized positive charge combined with disruption of the hydrogen-bonding network accounts for their inactivity. Together, these findings establish an essential role for R512 in catalytic activity and suggest how mutation of this residue can disrupt the BER pathway and contribute to tumorigenesis.

This work demonstrates successful grafting of N-hydroxyethyl acrylamide (HEAA) onto poly[(R)-3-hydroxybutyrate] (P[(R)-3HB]), up to 156 wt.% using [60]Co gamma irradiation (10.1 kGy) and a 1:4 P[(R)-3HB]:HEAA molar ratio in ethyl acetate. The resulting N-hydroxyethyl acrylamide-grafted poly[(R)-3-hydroxybutyrate] (P3HB-HEAA) was comprehensively characterized using FTIR, NMR, SEM, DSC, TGA/DTG, tensile testing, and water uptake, and exhibited striking property changes relative to the native polymer. The modified material showed a Tg of 124°C, reduced melting enthalpy (ΔHm = 12 J/g), increased Td,10 and Td,50 of 260°C and 340°C, respectively, increased elongation at break (ɛ = 65%), increased toughness (by 378%), exceptional water absorption capacity (610 wt.%), a markedly less ordered surface structure compared to the starting materials. These changes indicate formation of a less crystalline, highly hygroscopic, hydrogel-like system. HEAA grafting generated a biphasic architecture comprising a semicrystalline, structurally robust, P[(R)-3HB] backbone coupled with an amorphous, hydrogel-like HEAA surface layer, yielding a potentially biodegradable polymer with enhanced hydration capacity. The structure retains structural integrity while enabling substantial water uptake properties not accessible with either component alone. Combining mechanical strength with tuneable hydration behavior, HEAA-grafted P[(R)-3HB] expands the functional and commercial potential of P[(R)-3HB] into applications requiring simultaneous structural integrity, controlled swelling, and potential biocompatibility.

Autophagy-related (ATG) proteins orchestrate canonical autophagy and related pathways, including LC3-associated phagocytosis (LAP). Autophagy and LAP play essential roles in immune homeostasis and regulation. Systemic sclerosis (SSc) is an autoimmune disease of unknown etiology characterized by progressive fibrosis affecting the skin and multiple internal organs, together with profound immune dysregulation. ATG alleles have been proposed as genetic susceptibility factors for SSc, suggesting that defects in autophagy-related pathways may contribute to the disease pathogenesis. We investigated whether canonical autophagy and/or LAP are altered in immune cells from patients with SSc. Autophagy was assessed by measuring LC3-II levels in circulating cells from SSc patients and controls, and autophagic flux was evaluated by flow cytometry following lysosomal inhibition. We then generated monocyte-derived macrophages and performed phagocytosis assays to assess the LAP activity. We evaluated the phagocytic capacity using IgG-coated beads that trigger LAP through Fcγ receptor engagement and measured the proportion of LC3-positive phagosomes (LAPosomes). No major defects in canonical autophagy were found in lymphocytes and monocytes isolated from the peripheral blood of patients with SSc. In contrast, monocyte-derived M0 macrophages from patients with SSc exhibited impaired LC3-associated phagocytosis. Impaired LAP in macrophages may contribute to the defective efferocytosis observed in SSc, thereby promoting persistent inflammation and fibrosis. Our findings identify LAP as a previously unrecognized pathway altered in SSc and suggest that therapeutic strategies aimed at restoring LAP activity may represent a novel approach for disease treatment.

GRAVITAS-119 (NCT03320642), a single-arm, open-label, phase 1, proof-of-concept study, assessed addition of itacitinib to standard-of-care graft-versus-host disease (GVHD) prophylaxis regimens in reduced-intensity conditioning peripheral blood allogeneic hematopoietic stem cell transplant. Day 28 hematologic recovery (percentage of patients demonstrating neutrophil recovery and platelet recovery) was the primary endpoint. Of 84 enrolled patients, 41 received itacitinib plus tacrolimus/methotrexate (nine with antithymocyte globulin [ATG]), 24 received itacitinib plus cyclosporine A/mycophenolate mofetil (16 with ATG), and 19 received itacitinib plus post-transplant cyclophosphamide/tacrolimus. Hematologic recovery was achieved by 51/55 (92.7%) evaluable patients (neutrophil recovery, 98.7%; platelet recovery, 94.6%). Estimated 1-year GVHD-free, relapse-free survival was 39.7%; estimated 1-year overall survival was 78.3%. Cumulative incidences for grade 2‒4 and grade 3‒4 acute GVHD at Day 180 were 13.2% and 4.8%, respectively; cumulative incidence of chronic GVHD at 1 year was 37.8%. Most common grade ≥3 adverse events were cytopenias; grade ≥3 infections occurred in 23 (27.4%) patients. Most patients receiving itacitinib plus standard-of-care GVHD prophylaxis achieved hematologic recovery, with any-grade GVHD and infection rates similar to previous reports with standard-of-care prophylaxis. Addition of itacitinib to GVHD prophylaxis permits timely hematologic recovery and does not increase GVHD or infection rates, although unmet need still remains for improved regimens.

High-resolution respirometry (HRR) of peripheral blood mononuclear cells (PBMCs) relies on controlled preanalytical procedures. Here, we present an optimized workflow for PBMC isolation from human whole blood, followed by short-term cryopreservation at -80°C. The protocol includes rapid thawing to preserve cell viability, washing steps to minimize platelet contamination, and quality-control assessment of PBMC number, viability, and composition before HRR measurements. Critical handling steps are optimized and validated by HRR against freshly prepared cells.

Allogeneic hematopoietic cell transplantation (allo‑HCT) has become increasingly feasible in older patients with hematologic malignancies, yet the optimal graft‑versus‑host disease (GvHD) prophylaxis in this population remains uncertain. We compared post‑transplant cyclophosphamide (PTCy) with anti‑thymocyte globulin (ATG) in patients aged ≥60 years undergoing reduced‑intensity conditioning (RIC) peripheral blood stem cell allo‑HCT, using EBMT registry data from 2016 to 2022. A total of 7507 patients were included: 1418 matched sibling donors (MSD), 5051 matched unrelated donors (MUD), and 1038 mismatched unrelated donor (MMUD) transplants. In MSD recipients aged >65 years, PTCy was associated with reduced relapse, improved progression‑free survival (PFS), and superior GvHD‑ and relapse‑free survival (GRFS). However, in MSD recipients aged 60-65 years, these outcomes did not differ significantly between PTCy and ATG. Among MUD recipients, PTCy was associated with lower relapse and chronic GvHD, and a higher PFS, GRFS, and overall survival. In MMUD transplants, outcomes were not significantly different. Our findings indicate that PTCy is a safe and effective alternative to ATG in older allo‑HCT recipients, especially in MUD transplants and in MSD recipients aged >65 years, but the findings should be interpreted cautiously given their observational nature and patient heterogeneity. Further studies are warranted to assess long‑term outcomes.

Autophagy is a conserved catabolic process essential for cellular homeostasis and adaptation to nutrient stress. The protozoan parasite Giardia lamblia lacks most canonical autophagy-related (ATG) genes, including the hallmark ATG8, raising longstanding questions about whether this deeply divergent parasite can perform autophagy. Here, we identify an ATG8-independent autophagy-like pathway in Giardia regulated by GlRac, the parasite's sole Rho family GTPase. GlRac-positive double-membrane compartments are induced by encystation and nutrient depletion, and their abundance rapidly declines following amino acid replenishment but is unaffected by glucose, indicating amino acid-specific regulation. Giardia Target of Rapamycin (GTOR) levels decrease during nutrient depletion, and GTOR knockdown increases compartment abundance, identifying GTOR as a negative regulator of compartment formation and linking this pathway to nutrient sensing. Time-lapse microscopy revealed that these compartments form through linear and cup-shaped intermediates before becoming spherical and are subsequently cleared upon nutrient replenishment. Of nine putative ATG orthologs examined, none localized as specifically as GlRac to these structures, supporting the existence of a highly divergent pathway. Nevertheless, the compartments exhibit multiple conserved autophagy-associated features, including double-membrane morphology, actin recruitment, acidification, and cysteine protease activity. Pharmacological inhibition of cysteine proteases with E-64d or blocking V-ATPase-mediated acidification with concanamycin A promotes compartment accumulation, consistent with continuous degradative turnover. GlRac regulates compartment biogenesis bidirectionally: constitutive activation increases compartment abundance and size, whereas knockdown reduces them. Finally, quinacrine, an FDA-approved antigiardial drug that accumulates in acidic organelles, perturbs GlRac-positive compartments, consistent with its reported effects on autophagy in other eukaryotes, raising the possibility that this pathway contributes to parasite fitness. Together, these findings establish GlRac as a central regulator of an ATG8-independent autophagy-like pathway in Giardia and demonstrate that this parasite retains key structural, regulatory, and degradation-associated features of autophagy despite the apparent absence of most canonical ATG machinery.

To validate the potential role of CCSER1, a candidate gene for litter size and yearling weight identified by selection signature analysis, in lambing performance and growth development of Junken meat sheep, CCSER1 polymorphisms were genotyped using the Sequenom MassARRAY[®] SNP assay. Association analysis was conducted to assess the associations of CCSER1 polymorphisms and SNP2-SNP3 diplotypes with growth and lambing-related traits, and reverse transcription quantitative PCR (RT-qPCR) was used to determine the expression profile of CCSER1 in the ewes' digestive, developmental and reproductive tissues. The results showed three missense mutations in the CCSER1 gene of Junken meat sheep were identified, including SNP1 (XM_042251242.2: c.2557C>T) in exon 11, SNP2 (XM_042251242.2: c.1206A>G) and SNP3 (XM_042251242.2: c.16T>C) in exon 2. Three SNPs were exploratorily predicted to alter the secondary and tertiary structures of the CCSER1 protein. A single-stage mixed model was applied to evaluate the associations between individual SNP genotypes and phenotypic variation, with Bonferroni correction subsequently performed for multiple testing within each trait. The results showed that the associations of SNP2 with ABW (adjusted P = 0.0024) and SNP3 with YW (adjusted P = 0.022) and ADG2 (adjusted P = 0.039) remained statistically significant after Bonferroni correction. Phasing analysis of SNP2 and SNP3 identified four chromosome-level haplotypes and six corresponding diplotypes. A significant diplotype association was observed for YW (adjusted P = 0.039). Tukey's HSD test indicated that individuals carrying the T-A/T-A or T-A/T-G diplotypes exhibited significantly higher YW compared with those carryingT-G/T-G diplotype. The RT-qPCR analysis revealed a tissue-specific expression profile of CCSER1, with the highest expression detected in the abomasum (P < 0.01). In addition, CCSER1 expression levels in the lung and ovary were significantly higher than those in the liver, kidney, skin, brain, and pituitary gland (P < 0.01). These findings suggest that CCSER1 is a candidate gene for growth and lambing-related traits in Junken meat sheep, and that SNP2, SNP3, T-A/T-A and T-A/T-G may serve as useful molecular markers for marker-assisted selection to improve lambing performance and growth development in sheep.

Microwave imaging is promising for non-contact inspection of radar-absorbing materials (RAMs); however, strong structured non-damage responses in reconstructed microwave images can resemble surface defects and trigger false detections, while hard masking may remove weak damage responses near ambiguous boundaries. To address these issues, this paper proposes dual-channel scale-adaptive clutter suppression with confidence-derived gating as an application-specific front end for surface-damage detection in curved RAM specimens. A normalized microwave-response map and a fixed morphology-enhanced map, both deterministically derived from the same measurement, are jointly encoded. The input-conditioned scale-adaptive pyramid-pooling module (SA-PPM) reweights multi-scale context branches, while confidence-derived adaptive-threshold gating (C-ATG) generates a bounded pixel-wise threshold from the retention confidence and its ambiguity proxy. Continuous-weight filtering (CW-FI) then produces continuous retention weights, enabling gradual attenuation instead of forced binary removal. Experiments were conducted on a paired visible-light/microwave RAM dataset. On the held-out test subset, the method achieved pixel-level discrimination between clutter and retained responses and improved detection of strip-like cracks and circular spalling under matched detector-side settings.

Traditional weight-based dosing results in variable rabbit anti-thymocyte globulin (rATG) clearance, delaying CD4+Tcell-immune-reconstitution (CD4 + IR) and impacting outcomes. In a retrospective pharmacokinetic/pharmacodynamic analysis of patients undergoing first T-replete hematopoietic cell transplantation (HCT), enrolled on BMT CTN 1202, we estimated post-HCT rATG-exposures as area under the curve (arbitrary unit per day/milliliter [AUxd/mL]) using a validated pharmacokinetic-model. Results were compared to patients who did not receive rATG. Previously defined post-HCT rATG-exposure groups: A: < 30, B:30-55 and C: ≥ 55 AUxd/mL were correlated with outcomes of interest using cox-proportional-hazard and cause-specific-hazards models. 325 patients (median age 51 years) were included: 228 received rATG. Median post-HCT rATG-exposure was 44.1 (Range 6.2-125.0). Among patients who received rATG, higher exposure correlated with worse five-year overall survival (OS) (no-ATG:51%; group A:67%; B:49%; C:34%, p = 0.01), higher five-year relapse incidence (no-ATG:31%; A:27%; B: 42%; C: 58%, p < 0.001) and lower CD4 + IR (no-ATG:64%; A:73%; B:51%; C:19%, p = 0.001). Grade 2-4 acute graft versus host disease (GVHD) rates were: no-ATG:40% ; A:26%, B:39%, C:35%, (p = 0.44). Any rATG-exposure was associated with lower moderate/severe chronic GVHD (HR:0.63, p = 0.025). Low post-HCT rATG-exposure (< 30AUxd/mL) but not absent correlated with higher OS, lower relapse, and lower GVHD related deaths. Model-based-dosing could be used to target optimal post-HCT rATG-exposure and improve HCT outcomes.