Relative Copy Research Articles

Overexpression of KPC contributes to ceftazidime-avibactam heteroresistance in clinical isolates of carbapenem-resistant Klebsiella pneumoniae

Ceftazidime–avibactam (CZA) is one of the effective antibiotics used for the treatment of carbapenem-resistant Klebsiella pneumoniae (CRKP) infections, but its resistance rate has increased recently. Previous studies have focused on the mechanisms of CZA resistance, while its heteroresistance in CRKP remains poorly understood. This study aimed to investigate the characteristics and mechanisms of CZA heteroresistance in CRKP isolates. A total of 311 CRKP clinical strains were collected in China from 2020 to 2022. The MICs of CZA and other antibiotics against K. pneumoniae were determined by broth microdilution method. The occurrence of CZA heteroresistance in CRKP was evaluated with population analysis profiling (PAP) and their characteristics were detected by polymerase chain reaction (PCR). The underlying mechanism of CZA heteroresistance in CRKP strains was investigated by molecular sequencing, whole genome sequencing (WGS), quantitative real-time PCR (qRT-PCR), and in vitro functional experiments. Strategies for preventing the emergence of CZA heteroresistance and alternative treatment options for strains exhibiting CZA heteroresistance were further explored. Thirty-four (12.4%) CZA-susceptible CRKP isolates were found to exhibit heteroresistance to CZA. All heteroresistant strains belonged to KPC-2 (97.1%) or KPC-3 (2.9%). The dominant multilocus sequence typing (MLST) was ST11 (64.7%) and the prevalent capsular serotypes were KL47 (38.2%) and KL64 (32.4%). Imipenem-relebactam and meropenem-vaborbactam still exhibited excellent antimicrobial activity against the resistant subpopulations of CZA heteroresistant strains. No significant mutations were found in KPC, OmpK35/36, PBP2/3, and LamB in resistant subpopulations. The relative expression and copy number of blaKPC were significantly upregulated in 47.1% and 35.3% of the resistant subpopulations compared with their parental strains, respectively. Silencing blaKPC expression significantly decreased the CZA MIC in resistant subpopulations with high blaKPC expression and hindered the emergence of CZA heteroresistance in their parental strains. Moreover, increasing the avibactam concentration to 8 or 16 mg/L or combining CZA with 0.5 × MIC tigecycline significantly suppressed the formation of CZA heteroresistance (P<0.05). In conclusion, we identified the occurrence of CZA heteroresistance in CRKP in China, which was attributed to the overexpression of KPC. Increasing the concentration of avibactam or combining CZA with tigecycline could effectively prevent the development of CZA heteroresistance in CRKP isolates. Besides, imipenem-relebactam and meropenem-vaborbactam may serve as alternative therapeutic options when clinical isolates with CZA heteroresistance are detected.

Organellar genome dynamics of exogenous stages of Eimeria tenella

BackgroundCoccidia are a group of intracellular protozoal parasites within the phylum Apicomplexa. Eimeria tenella, one of the species that cause intestinal coccidiosis in poultry, can cause significant mortality and morbidity. Diploid oocysts of Eimeria species are shed in the feces of an infected host and must sporulate to achieve infectivity. This process results in eight haploid infectious units, called sporozoites, held within a single oocyst. Each Eimeria spp. parasite possesses a single apicoplast and a single mitochondrion, both of which carry multiple copies of their respective organellar genomes. Reports of copy numbers of organellar genomes have varied widely.MethodsWe report the application of quantitative polymerase chain reaction (qPCR), supported by next-generation sequencing, for the quantification of the extranuclear genomes relative to the nuclear genome over the course of sporulation and following its completion.ResultsAt 64 elapsed hours, 93.0% of oocysts were fully sporulated; no increase in percent sporulation was observed after this time. Apicoplast relative genome copy number showed several significant shifts up to 72 elapsed hours, after which no significant shifts were observed. Oocysts were shed with approximately 60% the amount of apicoplast DNA present at 72 h, after which point no significant shifts in apicoplast genome relative abundance occurred. Mitogenome relative copy number showed only two significant shifts, from 16 to 24 elapsed hours and from 24 to 32 elapsed hours. Oocysts were shed with approximately 28% the amount of mitochondrial DNA that was present at the time sporulation was deemed morphologically complete, at 64 elapsed hours.ConclusionsThe characterization of the dynamics of genome abundance in exogenous stages sheds new light on the basic biology of Eimeria spp. and supports the use of extranuclear targets for molecular modes of parasite quantification and identification with improved sensitivity and accuracy.Graphical

PoxtA amplification and mutations in 23S rRNA confer enhanced linezolid resistance in Enterococcus faecalis.

This study aimed to explore the evolutionary patterns and resistance mechanisms of an Enterococcus faecalis strain harbouring poxtA under linezolid exposure. A poxtA-carrying E. faecalis electrotransformant DJH702 with a linezolid minimum inhibitory concentration of 4 mg/L was exposed to increasing concentrations of linezolid (8-64 mg/L). The derived strains growing at 8, 16, 32 and 64 mg/L, designed DJH702_8, DJH702_16, DJH702_32 and DJH702_64, were obtained. The amplification and overexpression of poxtA were measured using sequencing and RT-PCR, the fitness cost by competition assays and the stability of the repeat units by serial passage. In all derived strains, high-level linezolid resistance develops through poxtA amplification. The relative copy numbers and transcription levels of poxtA were significantly increased. However, in the presence of higher linezolid concentrations, DJH702_32 and DJH702_64 showed reduced poxtA copy numbers and transcription levels compared with DJH702_8 and DJH702_16, but additional mutations in the 23S rRNA (G2505A). IS1216E-mediated formation of translocatable units with subsequent tandem amplification of these translocatable units supported the gain of poxtA segments. However, these amplicons were not stable and were lost frequently in the absence of a linezolid selection pressure. The amplification of the poxtA region did not result in a fitness cost, but mutations in 23S rRNA did. poxtA-carrying E. faecalis electrotransformants used two distinct mechanisms to resist linezolid selection pressure: at lower concentrations, strains prioritized increasing poxtA expression levels, while at higher concentrations, a combination of increased poxtA expression and mutations in 23S rRNA was observed.

Effect of subinhibitory concentrations on the spreading of the ampicillin resistance gene blaCMY-2 in an activated sludge microcosm

ABSTRACT As the problem of multi-resistant bacteria grows a better understanding of the spread of antibiotic resistance genes is of utmost importance for society. Wastewater treatment plants contain subinhibitory concentrations of antibiotics and are thought to be hotspots for antibiotic resistance gene propagation. Here we evaluate the influence of sub-minimum inhibitory concentrations of antibiotics on the spread of resistance genes within the bacterial community in activated sludge laboratory-scale sequencing batch reactors. The mixed communities were fed two different ampicillin concentrations (500 and 5000 µg/L) and the reactors were run and monitored for 30 days. During the experiment the β-lactamase resistance gene bla CMY-2 was monitored via qPCR and DNA samples were taken to monitor the effect of ampicillin on the microbial community. The relative copy number of bla CMY-2 in the reactor fed with the sub-minimum inhibitory concentration of 500 µg/L ampicillin was spread out over a wider range of values than the control and 5000 µg/L ampicillin reactors indicating more variability of gene number in the 500 µg/L reactor. This result emphasises the problem of sub-minimum inhibitory concentrations of antibiotics in wastewater. High-throughput sequencing showed that continuous exposure to ampicillin caused a shift from a Bacteroidetes to Proteobacteria in the bacterial community. The combined use of qPCR and high-throughput sequencing showed that ampicillin stimulates the spread of resistance genes and leads to the propagation of microbial populations which are resistant to it.

Comparison of Rodent Infectious Agent Detection by Exhaust Dust Testing and Traditional Sentinel Testing Using Quantitative Polymerase Chain Reaction.

Improved diagnostic capabilities and a desire to reduce or refine the use of animals as soiled bedding sentinels (SBS) have driven interest in developing the use of PCR-based testing methods, such as exhaust dust testing (EDT), for routine rodent health surveillance. We compared the absolute and quantitative PCR results from EDT filters with SBS mice by routine screening via a panel of 19 infectious agents including agents known to be excluded or present in the colony. In this study, EDT and SBS were compared at days 0, 90, and 180 in 3 facilities (n = 12 rooms) with animals housed on IVC racks (n = 19 double-sided and 23 single-sided racks). All racks were negative for excluded agents (n = 15 agents) during the study. The bacterial agent Helicobacter spp. was consistently detected on EDT filters while less consistently detected via SBS. EDT filters detected Corynebacterium bovis better than SBS in areas where the agent was present. EDT filters and SBS mice tested for murine norovirus (MNV) demonstrated agreement for positive tests by both PCR and serology. For rodent chaphamaparvovirus-1 (RCHPV-1) we compared EDT to urine and feces from SBS. Six cages of SBS were positive for RCHPV-1 by fecal PCR with 5 out of 6 testing positive on urine, while only 3 out of 6 EDT filters tested positive. Since real-time fluorogenic PCR was used for testing, relative PCR copy numbers for each positive finding were evaluated to estimate organism load at the rack level. Copy numbers allowed for further characterization of agent presence within a colony. Furthermore, we compared copy numbers with cage census for MNV and Helicobacter spp., which was positively correlated for EDT testing but not for SBS. Overall, our results demonstrate that EDT's ability to detect many commonly excluded agents is comparable to or better than SBS.

Characterization and functional insights of the novel RC-type plasmid pAnox1 from Anoxybacillus gonensis 05S15

The plasmid pAnox1, isolated from Anoxybacillus gonensis 05S15, was sequenced and characterized as a circular, double-stranded DNA molecule of 1592 base pairs with a GC content of 40.01 %. Despite its cryptic nature and small genome, bioinformatic analyses identified conserved motifs associated with replication-related proteins, though BLAST searches revealed no significant homology with other plasmids. The plasmid genome contains five putative Open Reading Frames (ORFs), four palindromic sequences, and two direct repeats on both strands, suggesting regulatory roles. Electron microscopy and Southern hybridization studies confirmed that pAnox1 follows a Rolling Circle (RC) replication mode. The study further demonstrated that the plasmid encodes three distinct transcripts: ORF-1 and ORF-3 are oriented in the same direction, while ORF-5 is on the opposite strand. RACE and LACE analyses revealed transcript lengths of 903 bp for ORF1, 499 bp for ORF3, and 211 bp for ORF5. Quantitative real-time PCR estimated the relative copy number of pAnox1 at 127 ± 2 copies per chromosomal equivalent. This novel RC-type plasmid in the Anoxybacillus genome holds promise as a cloning and expression vector for biotechnological applications and in vivo protein engineering.

Transfer of antibiotic resistance genes from soil to rice in paddy field

The global spread and distribution of antibiotic resistance genes (ARGs) has received much attention whereas knowledge about the transmission of ARGs from one matrix to another is still insufficient. In this study, the paddy fields fertilized with chemical fertilizer, swine compost, and no fertilizer were investigated to assess the transfer of ARGs from soil to rice. Soil and plant samples were collected at day 0, 7, 30 and 79 representing various stages of paddy growth. High throughput qPCR was applied to quantify ARGs using a set of 144 primers. Gene copy number of ARGs measured in soil initially decreased and then increased in soil with no fertilizer and chemical fertilizer, indicating that crop planting and flooding conditions did influence the ARGs profiles in soil. Application of swine compost significantly enhanced the relative abundance and gene copy number of ARGs in paddy soil. Rice seedlings contained substantial amount of ARGs and their relative abundance continually decreased after transplant. Compared with initial stage, detection frequencies of ARGs increased in soil without swine compost at harvest time (day 79), indicating the transmission of ARGs from irrigation water to soil. Detection frequencies of ARGs increased in soil and rice root with swine compost at harvest time, indicating the transfer of ARGs from swine compost to soil and rice root. There was no significant difference in abundance and diversity of ARGs in rice grains with these three different fertilizations. The source of the ARGs in rice grain still needs further exploration.

Mitochondrial DNA copy number and risk of papillary thyroid carcinoma

BackgroundMitochondrial DNA (mtDNA) copy number is associated with tumor activity and carcinogenesis. This study was undertaken to investigate mtDNA copy number in papillary thyroid cancer (PTC) tissues and to evaluate the risk of PTC development. The clinicopathological features of patients and mtDNA copy number were correlated. The value of mtDNA copy number was evaluated as a biomarker for PTC.MethodDNA was extracted from 105 PTC tissues and 67 control thyroid tissues, and mtDNA copy number mtDNA oxidative damage were determined using qPCR techniques.ResultsOverall, the relative mtDNA copy number was significantly higher in PTC patients (p < 0.001). The risk of developing PTC increased significantly across the tertiles of mtDNA copy number (p trend < 0.001). The higher the mtDNA copy number tertile, the greater the risk of developing PTC. Patients with follicular variants had an odds ratio of 2.09 (95% CI: 1.78–2.44) compared to those with classical variants (p < 0.001). The level of mtDNA oxidative damage in PTC was significantly elevated compared to controls (p < 0.001). The ROC analysis of mtDNA copy number indicated an area under the curve (AUC) of 77.7% (95% CI: 0.71 to 0.85, p < 0.001) for the ability of mtDNA copy number z-scores in differentiate between PTC and controls.ConclusionOur results indicated that the augmentation of mtDNA content plays a significant role during the initiation of thyroid cancer, and it might represent a potential biomarker for predicting the risk of PTC.

Relative rDNA copy number is not associated with resistance training-induced skeletal muscle hypertrophy and does not affect myotube anabolism in vitro.

Ribosomal DNA (rDNA) copies exist across multiple chromosomes and inter-individual variation in copy number is speculated to influence the hypertrophic response to resistance training. Thus, we examined if rDNA copy number was associated with resistance training-induced skeletal muscle hypertrophy. Participants (n=53 males, 21±1 years old; n=29 females, 21±2 years old) performed 10-12 weeks of full-body resistance training. Hypertrophy outcomes were determined, as was relative rDNA copy number from pre-intervention vastus lateralis (VL) biopsies. Pre- and post-intervention VL biopsy total RNA was assayed in all participants, and mRNA/rRNA markers of ribosome content and biogenesis were also assayed in the 29 females prior to training, 24 hours following training bout 1, and in the basal state after 10 weeks of training. Across all participants, no significant associations were evident between relative rDNA copy number and training-induced changes in whole body lean mass (r = -0.034, p=0.764), vastus lateralis thickness (r = 0.093, p=0.408), mean myofiber cross-sectional area (r = -0.128, p=0.259), or changes in muscle RNA concentrations (r = 0.026, p=0.818), and these trends were similar when examining each gender. However, all Pol-I regulon mRNAs as well as 45S pre-rRNA, 28S rRNA and 18S rRNA increased 24 hours following the first training bout in females. Follow-up studies using LHCN-M2 myotubes demonstrated a reduction in relative rDNA copy number induced by bisphenol A (BPA) did not significantly affect insulin-like-growth factor-induced myotube hypertrophy. These findings suggest relative rDNA copy number is not associated with myofiber hypertrophy.

The genomic trajectory of ovarian high-grade serous carcinoma can be observed in STIC lesions.

Ovarian high-grade serous carcinoma (HGSC) originates in the fallopian tube, with secretory cells carrying a TP53 mutation, known as p53 signatures, identified as potential precursors. p53 signatures evolve into serous tubal intraepithelial carcinoma (STIC) lesions, which in turn progress into invasive HGSC, which readily spreads to the ovary and disseminates around the peritoneal cavity. We recently investigated the genomic landscape of early- and late-stage HGSC and found higher ploidy in late-stage (median 3.1) than early-stage (median 2.0) samples. Here, to explore whether the high ploidy and possible whole-genome duplication (WGD) observed in late-stage disease were determined early in the evolution of HGSC, we analysed archival formalin-fixed paraffin-embedded (FFPE) samples from five HGSC patients. p53 signatures and STIC lesions were laser-capture microdissected and sequenced using shallow whole-genome sequencing (sWGS), while invasive ovarian/fallopian tube and metastatic carcinoma samples underwent macrodissection and were profiled using both sWGS and targeted next-generation sequencing. Results showed highly similar patterns of global copy number change between STIC lesions and invasive carcinoma samples within each patient. Ploidy changes were evident in STIC lesions, but not p53 signatures, and there was a strong correlation between ploidy in STIC lesions and invasive ovarian/fallopian tube and metastatic samples in each patient. The reconstruction of sample phylogeny for each patient from relative copy number indicated that high ploidy, when present, occurred early in the evolution of HGSC, which was further validated by copy number signatures in ovarian and metastatic tumours. These findings suggest that aberrant ploidy, suggestive of WGD, arises early in HGSC and is detected in STIC lesions, implying that the trajectory of HGSC may be determined at the earliest stages of tumour development. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Dam construction accelerated the development of biological soil crusts in degraded soil patches in the Lhasa River basin, Qinghai-Tibetan Plateau

The Lhasa River Basin currently faces severe land degradation. In recent years, several dams have been built on the main stream, whereas, their impact on land desertification remains less elucidated. Herein, we investigated variances of soil properties and microbial communities in biological soil crusts (BSCs) along the valley area of Lhasa River and the mountain slope (altitude gradient) in reservoir area after five years of dam construction. Soil nutrient and water contents, enzyme activities, and N-cycle related functional gene copies significantly increased in reservoir-affected areas and decreased with the increase of mountain slope altitude and the distance away from the reservoir, which were usually degraded soil patches; similarly, the relative abundance of bacteria, fungi and moss in BSCs significantly increased in reservoir-affected areas, whilst that of cyanobacteria was higher in degraded soil patches. The linear and structural equation models also showed that dam construction altered water distribution status of soil surface, which enhanced soil properties, N-cycle and increased the relative abundance of moss of BSCs, and successfully accelerated the development and succession of BSCs in degraded soil. This study firstly reported that dam construction could accelerate the succession process of degraded soil in the reservoir affected area and thus a reference for ecological restoration of desertification soil of the Lhasa River Basin in QTP.

A Preliminary Study on the Identification of Genes Involved in Lignification in the Endocarp of Bared-Nut Walnut (Juglans regia L.) in Xinjiang, China

This study focused on the “Xinlu” walnut and explored the molecular regulatory mechanism of lignin synthesis in the endocarp, aiming to explain the formation of bared-nut walnuts through morphological, metabolomic, and transcriptomic techniques. It was found that the synthesis of lignin, cellulose, p-coumaryl alcohol, and sinapyl alcohol was severely inhibited in the pulpy-hue (PUH) of the endocarp. We obtained 14 modules (gene sets) significantly correlated with the lignification factor (LIG) and 1548 hub genes. Additionally, we identified a MEplum3 module involved in endocarp lignin synthesis, primarily participating in phenylalanine biosynthesis and the lignin biosynthetic process. Meanwhile, we constructed a gene co-expression network for the MEplum3 module and identified a key hub gene for lignin synthesis—JrCAD10. Among the different tissues of “Xinlu”, the expression level of JrCAD10 in the scleritic-hue (SCH) was significantly higher than in other tissues, with a relative copy number (RCN) of 3.2. However, JrCAD10 expression was severely suppressed in the PUH. The suppression of JrCAD10 expression led to the inhibition of lignin monomer synthesis, which further resulted in inhibited lignin synthesis, thus forming the bared-nut walnut. Our findings provide new insights into understanding the regulation of lignin synthesis and offer a possible explanation for the formation of bared-nut walnuts.

BDNF/TrkB Induces pCREBS133 Phosphorylation and Activates Mitochondrial Biogenesis in Rat Phrenic Motor Neurons

Neural control of the diaphragm muscle (DIAm) is essential to sustain breathing. During breathing, fatigue resistant DIAm motor units are recruited comprising smaller phrenic motor neurons (PhMNs) and type I and IIa muscle fibers. Larger PhMNs comprise more fatigable DIAm motor units that are infrequently active during expulsive behaviors. The difference in activation history is reflected in the higher mitochondrial volume density in smaller PhMNs. In PhMNs, brain-derived neurotrophic factor (BDNF) signals through its high-affnity receptor, tropomyosin receptor kinase B (TrkB.FL). Previously, we found that intrathecal BDNF treatment induces CREB phosphorylation at Serine 133 (pCREBs133). We hypothesized that pCREBs133 binds to the PGC1a promoter, transcriptionally targeting PGC1a expression, mediating downstream increased expression of NRF1, NRF2, and TFAM and mitochondrial biogenesis. To test this hypothesis, we used a chemosensitive TrkBF616 rat model with a knock-in allele sensitive to 1NMPP1 in that TrkB kinase is inhibited following 1NMPP1, a small molecule derivative of the protein kinase inhibitor protein phosphatase 1 that plays a significant role inhibiting the TrkB phosphorylation and activation. Systemic treatment of 1NMPP1 in TrkBF616 rats inhibits TrkB kinase activity. A bioinformatic analysis was performed to identify a putative binding site for pCREBs133 in the PGC1a promoter sequence in rats. Using a chromatin immunoprecipitation (ChIP) assay we found that pCREBS133 binds to the PGC1a promoter in rat cervical ventral horn. We showed that intrathecal BDNF treatment increased pCREBs133 phosphorylation, which was blocked by 1NMPP1 treatment in TrkBF616 rats. PGC1a, NRF1/2, and TFAM protein was compared in cervical ventral horn lysates from BDNF- and BDNF+1NMPP1-treated TrkBF616 rats by qRT-PCR and Western Blot. Relative mtDNA copy number (normalized to nuclear DNA) was quantified by qPCR to assess the mitochondrial biogenesis. All results were analyzed by paired t-test. Following 1NMPP1-induced inhibition of TrkB kinase, PGC1a, NRF1/2, and TFAM mRNA and protein expression was reduced. Consistent with these findings, mtDNA copy number was decreased following TrkB kinase inhibition by 1NMPP1. We conclude that inhibition of TrkB kinase by 1NMPP1 reduces PGC1a-mediated mitochondrial biogenesis in rat cervical ventral horn. Supported by NIH grants AG44615 and HL146114. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Proteomics Reveals the Obstruction of Cellular ATP Synthesis in the Ruminal Epithelium of Growth-Retarded Yaks.

Growth-retarded yaks are of a high proportion on the Tibetan plateau and reduce the economic income of farmers. Our previous studies discovered a maldevelopment in the ruminal epithelium of growth-retarded yaks, but the molecular mechanisms are still unclear. This study aimed to reveal how the proteomic profile in the ruminal epithelium contributed to the growth retardation of yaks. The proteome of the ruminal epithelium was detected using a high-resolution mass spectrometer. There were 52 proteins significantly differently expressed between the ruminal epithelium of growth-retarded yaks and growth-normal yaks, with 32 downregulated and 20 upregulated in growth-retarded yaks. Functional analysis showed the differently expressed proteins involved in the synthesis and degradation of ketone bodies (p = 0.012), propanoate metabolism (p = 0.018), pyruvate metabolism (p = 0.020), and mineral absorption (p = 0.024). The protein expressions of SLC26A3 and FTH1, enriched in the mineral absorption, were significantly downregulated in growth-retarded yaks. The key enzymes ACAT2 and HMGCS2 enriched in ketone bodies synthesis and key enzyme PCCA enriched in propanoate metabolism had lower protein expressions in the ruminal epithelium of growth-retarded yaks. The ATP concentration and relative mitochondrial DNA copy number in the ruminal epithelium of growth-normal yaks were dramatically higher than those of growth-retarded yaks (p < 0.05). The activities of citrate synthase (CS), the α-ketoglutarate dehydrogenase complex (α-KGDHC), isocitrate dehydrogenase (ICD) in the tricarboxylic acid cycle (TCA), and the mitochondrial respiratory chain complex (MRCC) were significantly decreased in ruminal epithelium of growth-retarded yaks compared to growth-normal yaks (p < 0.05). The mRNA expressions of COQ9, COX4, and LDHA, which are the encoding genes in MRCC I, IV and anaerobic respiration, were also significantly decreased in the ruminal epithelium of growth-retarded yaks (p < 0.05). Correlation analysis revealed that the average daily gain (ADG) was significantly positively correlated to the relative mitochondrial DNA copy number (p < 0.01, r = 0.772) and ATP concentration (p < 0.01, r = 0.728) in the ruminal epithelium, respectively. The ruminal weight was positively correlated to the relative mitochondrial DNA copy number (p < 0.05, r = 0.631) and ATP concentration in ruminal epithelium (p < 0.01, r = 0.957), respectively. The ruminal papillae had a significant positive correlation with ATP concentration in ruminal epithelium (p < 0.01, r = 0.770). These results suggested that growth-retarded yaks had a lower VFA metabolism, ketone bodies synthesis, ion absorption, and ATP synthesis in the ruminal epithelium; it also indicated that the growth retardation of yaks is related to the obstruction of cellular ATP synthesis in rumen epithelial cells.

Tandem amplification of a plasmid-borne tet(A) variant gene confers tigecycline resistance in Escherichia coli.

To elucidate the mechanism of tigecycline resistance in Escherichia coli that is mediated by the tet(A) variant gene. E. coli strain 573 carried a plasmid-borne tet(A) variant gene, tentatively designated tet(A)TIG, that conferred decreased tigecycline susceptibility (MIC 0.5 mg/L). When exposed to increasing concentrations of tigecycline (0.25-8 mg/L), mutants growing at 2, 4 and 8 mg/L were obtained and sequenced. Copies of plasmid and tet(A)TIG relative to the chromosomal DNA in the mutants were determined by WGS and quantitative PCR (qPCR). Expression of tet(A)TIG in the mutants was evaluated by RT-qPCR. The tet(A)TIG-carrying plasmids were visualized by S1-PFGE and Southern blot hybridization. PCR served for the detection of a tet(A)TIG-carrying unconventional circularizable structure (UCS). Tigecycline resistance with maximum MICs of 16 mg/L was seen in E. coli mutants selected in the presence of tigecycline. Compared with the parental strain, the relative copy number and transcription level of tet(A)TIG in the mutants increased significantly in the presence of 2, 4 and 8 mg/L tigecycline, respectively. With increasing tigecycline selection pressure, the tet(A)TIG-carrying plasmids in the mutants increased in size, correlating with the number of tandem amplificates of a ΔTnAs1-flanked UCS harbouring tet(A)TIG. These tandem amplificates were not stable in the absence of tigecycline. Tigecycline resistance is due to the tandem amplification of a ΔTnAs1-flanked tet(A)TIG-carrying plasmid-borne segment in E. coli. The gain/loss of the tandem amplificates in the presence/absence of tigecycline represents an economic way for the bacteria to survive in the presence of tigecycline.

Abstract 3317: KRASmulti inhibitor BI 3706674 shows efficacy in KRAS-driven preclinical models of cancer that supports clinical testing in patients with tumors harbouring KRASG12V mutations and KRAS wild-type amplifications

Abstract Alterations in KRAS are a main cancer driver. Amplifications of the KRAS wild-type (wt) allele account for ~7% of all KRAS-driven cancers and are frequent in gastric (~5%), esophageal (~13%) and gastroesophageal junction cancers (~12%). KRASG12V mutations account for ~28% of pancreatic cancers, 9% of colorectal cancers and 6% of lung adenocarcinoma. There is an urgent medical need to identify targeted therapies for these frequent KRAS alterations. Herein, we describe BI 3706674, a novel, potent and orally bioavailable small molecule inhibitor of the KRAS oncogene for clinical testing in patients with tumors harboring KRASG12V mutations and KRAS wild-type amplifications. BI 3706674 binds non-covalently to multiple KRAS mutant alleles, including the KRAS wt allele, in the GDP-bound state and thereby disrupts oncogenic signaling. Importantly, BI 3706674 is highly selective for KRAS vs HRAS or NRAS, and it is therefore expected to be well-tolerated in normal tissues. In two large cancer cell line panels (PRISM and Horizon), BI 3706674 shows sensitivity across a wide range of KRAS alleles (KRAS wt amplifications and KRAS mutations e.g., G12A/C/D/V, G13D and Q61H). The strongest sensitivity was observed for cell lines with KRAS wt amplifications (relative copy number (CN) of &amp;gt; 10) followed by cell lines with KRASG12V mutations along with significant pharmacodynamic (PD) biomarker modulation (e.g., down-regulation of DUSP6 mRNA). In vivo, BI 3706674 was well-tolerated and showed dose-dependent efficacy in cell line-derived and patient-derived xenograft models of human gastric cancer with KRAS wt CN &amp;gt; 10 and diverse tumor types with KRASG12V mutations. A twice daily oral dose of 30 mg/kg induced significant tumor regression and PD biomarker modulation. Combination of BI 3706674 with standard of care therapies and novel agents are being tested in preclinical models to guide clinical development. Results of the ongoing pre-clinical analysis will be shared. A Phase I clinical trial is in preparation in patients with advanced solid cancers harboring KRASG12V mutations and KRAS wt amplifications to evaluate safety, tolerability, pharmacokinetic and pharmacodynamic properties, and efficacy of BI 3706674. Citation Format: Antonio Tedeschi, David H. Peng, Fiorella Schischlik, Lorenz Herdeis, Otmar Schaaf, Valeria Santoro, Daniel Gerlach, Fabio Savarese, Jesse Lipp, Christian Haslinger, Francesca Rocchetti, Johannes Popow, Heinrich J. Huber, Birgit Wilding, Matthias Treu, Julian Fuchs, Joachim Broeker, Tobias Wunber, Michael Gmachl, Klaus Rumpel, Matthew Rees, Melissa Ron, Jennifer Roth, Mariah Williams, Charles Deckard, Vandhana Ramamoorthy, Joseph R. Daniele, Jaffer A. Ajani, Funda Meric-Bernstam, Scott Kopetz, Michael Kim, Don L. Gibbons, Christopher P. Vellano, Joseph R. Marszalek, Timothy P. Heffernan, Darryl McConnell, Mark Pearson, Norbert Kraut, Dorothea Rudolph. KRASmulti inhibitor BI 3706674 shows efficacy in KRAS-driven preclinical models of cancer that supports clinical testing in patients with tumors harbouring KRASG12V mutations and KRAS wild-type amplifications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3317.

Mitochondrial Deoxyribonucleic Acid Copy Number Elevation As a Predictor for Extended Survival and Favorable Outcomes in High-Grade Brain Tumor Patients: A Malaysian Study.

Investigating the role of mitochondrial DNA (mtDNA) alterations and their impact on brain tumor progression remains a significant focus in cancer research. The research aimed to explore the specific contributions of mtDNA copy number changes and their correlations with patient survival, large mtDNA deletions, and TFAM mutations in brain tumor patients. A total of 41 patients with confirmed brain tumors underwent DNA extraction from both tumor tissues and blood samples. The relative mtDNA copy number in comparison to the nuclear genome was quantified using quantitative real-time polymerase chain reaction (qRT-PCR). Long-range PCR assessed largescale mtDNA deletions, and Sanger sequencing was applied to detect exon 4 TFAM mutations. Analysis revealed significantly increased mtDNA copy numbers in brain tumor tissues (80.5%) compared to matched blood samples (P < .001). Median delta Ct (∆Ct) values were 7.35 in cancerous tissues and 11.81 in blood (P <.001), with median relative mtDNA content of 0.0123 and 0.0006, respectively (P <.001). Patients with higher mtDNA copy numbers experienced longer overall survival periods (P=.045) and notably favorable outcomes, particularly in high-grade tumor cases (P=.016). Furthermore, a singlenucleotide deletion was identified in exon 4 of TFAM in a patient with glioblastoma IV, while no large-scale mtDNA deletions were found in brain tumor patients. Our study strongly supports the role of increased mtDNA copy numbers as a reliable predictor for improved survival and positive outcomes in high-grade brain tumor patients.

Exploring the dynamics of antibiotic resistome on plastic debris traveling from the river to the sea along a representative estuary based on field sequential transfer incubations

The environmental risks arising from ubiquitous microplastics or plastic debris (PD) acting as carriers of antibiotic resistance genes (ARGs) have attracted widespread attention. Enormous amounts of plastic waste are transported by rivers and traverse estuaries into the sea every year. However, changes in the antibiotic resistome within the plastisphere (the biofilms formed on PD) as PD travels through estuaries are largely unknown. In this study, we performed sequential migration incubations for PD along Haihe Estuary to simulate the natural process of PD floating from rivers to the ocean. Metagenomic sequencing and analysis techniques were used to track microbial communities and antibiotic resistome on migrating PD and in seawater representing the marine environment. The total relative gene copies of ARGs on traveling PD remained stable. As migration between greatly varied waters, additional ARG subtypes were recruited to the plastisphere. Above 80 % ARG subtypes identified in the plastisphere were persistent throughout the migration, and over 30 % of these persistent ARGs were undetected in seawater. The bacterial hosts composition of ARGs on PD progressively altered as transported downstream. Human pathogenic bacteria carrying ARGs (HPBs-ARG) exhibited decreasing trends in abundance and species number during transfer. Individual HPBs-ARG persisted on transferred PD and were absent in seawater samples, comprising Enterobacter cloacae, Klebsiella pneumoniae, Mycobacterium tuberculosis, and Vibrio parahaemolyticus. Based on all detected ARGs and HPBs-ARG, the Projection Pursuit model was applied to synthetically evaluate the potential risks of antibiotic resistance on migrating PD. Diminished risks on PD were observed upon the river-to-sea journey but consistently remained significantly higher than in seawater. The potential risks posed to marine environments by drifting PD as dispersal vectors for antibiotic resistance deserve greater attention. Our results provide initial insights into the dynamics or stability of antibiotic resistome on PD crossing distinct aquatic systems in field estuaries.

Assessment of Y chromosome copy number alterations in non-neoplastic and neoplastic leukocytes of male dogs

The loss of the Y chromosome (ChrY), also known as LOY, is a common genetic alteration observed in men. It occurs in non-neoplastic cells as an age-related change as well as in neoplastic cells of various cancer types. While well-documented in humans, LOY has not been extensively studied in non-human mammals. In this study, we developed simple digital PCR-based assays to assess the copy number of ChrY relative to the X chromosome (ChrX) and chromosome 8 (Chr8) to evaluate ChrY numerical alterations in male canine DNA specimens. Using these assays, we analyzed non-neoplastic leukocytes from 162 male dogs without hematopoietic neoplasia to investigate the occurrence of age-related LOY in non-neoplastic leukocytes. Additionally, we examined 101 tumor DNA specimens obtained from male dogs diagnosed with various types of lymphoma and leukemia to determine whether copy number alterations of the ChrY occur in canine hematopoietic cancers. Analysis of the 162 non-neoplastic leukocyte DNA specimens from male dogs of varying ages revealed a consistent ∼1:1 ChrY:ChrX ratio. This suggests that age-related LOY in non-neoplastic leukocytes is rare or absent in dogs. Conversely, a decreased or increased ChrY:ChrX ratio was detected in canine neoplastic leukocytes at varying frequencies across different canine hematopoietic malignancies (P = 0.01, Fisher's exact test). Notably, a higher incidence of LOY was observed in more aggressive cancer types. To determine if this relative LOY to ChrX was caused by changes in ChrY or ChrX, we further analyzed their relative copy numbers using Chr8 as a reference. Loss of ChrX relative to Chr8 was found in 21% (9/41) of B-cell lymphomas and 6% (1/18) of non-T-zone/high-grade T-cell lymphomas. In contrast, a subset (29%, 4/14) of T-cell chronic lymphocytic leukemia showed gain of ChrX relative to Chr8. Notably, no relative LOY to Chr8 was detected indolent hematopoietic cancers such as T-zone lymphoma (0/9) and chronic lymphocytic leukemia of B-cell (0/11) and T-cell origins (0/14). However, relative LOY to Chr8 was present in more aggressive canine hematopoietic cancers, with incidences of 24% (10/41) in B-cell lymphoma, 44% (8/18) in non-T-zone/high-grade T-cell lymphoma, and 75% (6/8) in acute leukemia. This study highlights both similarities and differences in LOY between human and canine non-neoplastic and neoplastic leukocytes. It underscores the need for further research into the role of ChrY in canine health and disease, as well as the significance of LOY across various species.

Toxic metals and essential trace elements in placenta and their relation to placental function

IntroductionPlacental function is essential for fetal development, but it may be susceptible to malnutrition and environmental stressors. ObjectiveTo assess the impact of toxic and essential trace elements in placenta on placental function. MethodsToxic metals (cadmium, lead, mercury, cobalt) and essential elements (copper, manganese, zinc, selenium) were measured in placenta of 406 pregnant women in northern Sweden using ICP-MS. Placental weight and birth weight were obtained from hospital records and fetoplacental weight ratio was used to estimate placental efficiency. Placental relative telomere length (TL) and mitochondrial DNA copy number (mtDNAcn) were determined by quantitative PCR (n = 285). Single exposure-outcome associations were evaluated using linear or spline regression, and joint associations and interactions with Bayesian kernel machine regression (BKMR), all adjusted for sex, maternal smoking, and age or BMI. ResultsMedian cadmium, mercury, lead, cobalt, copper, manganese, zinc, and selenium concentrations in placenta were 3.2, 1.8, 4.3, 2.3, 1058, 66, 10626, and 166 μg/kg, respectively. In the adjusted regression, selenium (>147 μg/kg) was inversely associated with placental weight (B: −158; 95 % CI: −246, −71, per doubling), as was lead at low selenium (B: −23.6; 95 % CI: −43.2, −4.0, per doubling). Manganese was positively associated with placental weight (B: 41; 95 % CI: 5.9, 77, per doubling) and inversely associated with placental efficiency (B: −0.01; 95 % CI: −0.019, −0.004, per doubling). Cobalt was inversely associated with mtDNAcn (B: −11; 95 % CI: −20, −0.018, per doubling), whereas all essential elements were positively associated with mtDNAcn, individually and joint. ConclusionAmong the toxic metals, lead appeared to negatively impact placental weight and cobalt decreased placental mtDNAcn. Joint essential element concentrations increased placental mtDNAcn. Manganese also appeared to increase placental weight, but not birth weight. The inverse association of selenium with placental weight may reflect increased transport of selenium to the fetus in late gestation.