Silent Mutations Research Articles

Genomic architecture and evolutionary relationship of BA.2.75: A Centaurus subvariant of Omicron SARS-CoV-2.

In this study, we explored the genomic architecture and phylogenomic relationship of BA.2.75, a subvariant of Omicron SARS-CoV-2. A set of 1468 whole-genome sequences of BA.2.75, submitted by 28 countries worldwide were retrieved from GISAID and used for finding genomic mutations. Moreover, the phylogenetic analysis of BA.2.75 was performed by using 2948 whole-genome sequences of all sub-variants of Omicron along with the Delta variant of SAS-CoV-2. We detected 1885 mutations, which were further grouped into 1025 missense mutations, 740 silent mutations, 72 mutations in non-coding regions, 16 in-frame deletions, 02 in-frame insertions, 8 frameshift deletions, 8 frameshift insertions and 14 stop-gained variants. Additionally, we also found 11 characteristic mutations having a prevalence of 81-99% and were not observed in any of the previously reported variant of SARS-CoV-2. Out of these mutations K147E, W152R, F157L, E210V, V213G, G339H were found in the NTD, and G446S & N460K in the RBD region of the Spike protein, whereas S403L and T11A were present in the NSP3, and E protein respectively. The phylogenetic relationship of this variant revealed that BA.2.75 is descended from the Omicron sub-variant BA.5. This evolutionary relationship suggests that the surge of BA.5 infections can reduce the severity of the infections accredited to BA.2.75. These findings would also improve our knowledge and understanding that how genetic similarities in different variants of SARS-CoV-2 can prime the immune system to fight off the infection caused by one subvariant, after defeating the other.

Identifikasi Mutasi Gen kelch 13 Penanda Resistensi Pada Plasmodium falciparum Dengan Pengobatan ACT Setelah 3 Hari Di Manokwari Papua Barat

Malaria is almost found all over the world, including Indonesia. WHO data on malaria case transmission in 2020, namely 241 million and 627,000 deaths. Female Anopheles mosquitoes are the main factor in the transmission of parasites, one of which is Plasmodium falciparum which is most dangerous to cause complications to death. Data from the Ministry of Health 2019 the highest transmission in Papua Province is 216,380 cases. The malaria elimination program uses ACT as a treatment therapy for malaria. The first appearance of ACT resistance in Cambodia was in 2008 to Plasmodium falciparum which was associated with the presence of a Kelch 13 gene mutation. The purpose of this study was to identify a mutation of the Kelch 13 gene markers of resistance in Plasmodium falciparum with ACT treatment after 3 days. This type of exploratory study used a population of all patients at the Amban Manokwari Health Center in West Papua, with a total sampling in August-September 2022. Of the 51 and 11 H3 samples met the inclusion criteria using RDT and Microscopic methods. The DNA isolated sample using Favorgen kit then amplified PCR and electrophoresis gel agarose. Then were 7 amplicons seen in the DNA band (±200bp) and confirmed results from PT. Science Genetics (±100bp), then 1 amplicon is continued for sequencing. The sequence results were analyzed using the Bioedit and Mega IX softwere with the alignment of the sequence results. Changes in nucleeid bases and amino acids were obtained so that the mutation of the Kelch 13 gene occurred, 3 mutation variants: substitution (transition & transversion), silent mutation (C3T codon 1 cysteine-cysteine), and missense mutation (T4A codon 2 tryptophan-serine). It can be concluded that the Plasmodium falciparum treatment ACT carriers the Kelch 13 gene mutation as one of the markers of resistance. Keywords: ACT, Plasmodium falciparum, Kelch 13 Gene Mutation.

Possible involvement of silent mutations in cancer pathogenesis and evolution

Recent studies have shown that some silent mutations can be harmful to various processes. In this study, we performed a comprehensive in silico analysis to elucidate the effects of silent mutations on cancer pathogenesis using exome sequencing data derived from the Cancer Genome Atlas. We focused on the codon optimality scores of silent mutations, which were defined as the difference between the optimality of synonymous codons, calculated using the codon usage table. The relationship between cancer evolution and silent mutations showed that the codon optimality score of the mutations that occurred later in carcinogenesis was significantly higher than of those that occurred earlier. In addition, mutations with higher scores were enriched in genes involved in the cell cycle and cell division, while those with lower scores were enriched in genes involved in apoptosis and cellular senescence. Our results demonstrate that some silent mutations can be involved in cancer pathogenesis.

Bacteria evade antibiotics with silent mutations to survive

Bacteria may develop antibiotic resistance due to unintentional DNA alterations that give them a survival advantage. Bacteria may develop antibiotic resistance due to unintentional DNA alterations that give them a survival advantage. Finding genetic changes and understanding how they enable bacteria to withstand antibiotic attacks will be crucial to our ability to develop new medications to combat them.

Evaluation of Xpert MTB/RIF Ultra assay for detection of Mycobacterium tuberculosis and rifampicin resistance

Tuberculosis (TB) is a public health challenge globally, and molecular testing is recommended to expedite diagnosis. Concerns that Xpert MTB/RIF assay (Xpert) may be less sensitive when testing paucibacillary samples led to the development of the Xpert MTB/RIF Ultra assay (Ultra). We evaluated the performance of Ultra against Xpert on clinical samples sent to the national reference laboratory in Singapore. In total, 149 samples collected between January 2019 and November 2020 were analysed. Mycobacterium tuberculosis complex (MTBC) was isolated from 55 cultures. Using culture as the reference standard, Ultra demonstrated higher sensitivity (96.4% vs 85.5%) and marginally lower specificity (88.3% vs 89.4%) compared to Xpert in the full cohort. When considering only paucibacillary specimens such as extrapulmonary and smear-negative samples, similar results were obtained. Reclassifying Ultra trace results (low levels of MTB are detected but no rifampicin resistant result is detected) as negative in the full cohort led to a decrease in sensitivity by 10.9% and a marginal increase in specificity by 1.1%. In instances of low bacillary load, Ultra also identified rifampicin resistance more accurately than Xpert, when corroborated against other methods such as broth microdilution, line probe assay and whole genome sequencing (WGS). One isolate tested rifampicin-resistant using Xpert and Ultra, but was phenotypically susceptible and WGS demonstrated the presence of the silent mutation Thr444Thr. Ultra is more sensitive than Xpert in the detection of MTBC and rifampicin resistance in our local setting. Nevertheless, the results of molecular testing should still be correlated with phenotypic studies.

Vkorc1 gene polymorphisms confer resistance to anticoagulant rodenticide in Turkish rats.

Mutations in Exon 1, 2 and 3 of the vitamin K epoxide reductase complex subunit 1 (Vkorc1) gene are known to lead to anticoagulant rodenticide resistance. In order to investigate their putative resistance in rodenticides, we studied the genetic profile of the Vkorc1 gene in Turkish black rats (Rattus rattus) and brown rats (Rattus norvegicus). In this context, previously recorded Ala21Thr mutation (R. rattus) in Exon 1 region, Ile90Leu mutation (R. rattus, R. norvegicus) in Exon 2 region and Leu120Gln mutation (R. norvegicus) in Exon 3 region were identified as "missense mutations" causing amino acid changes. Ala21Thr mutation was first detected in one specimen of Turkish black rat despite the uncertainty of its relevance to resistance. Ile90Leu mutation accepted as neutral variant was detected in most of black rat specimens. Leu120Gln mutation related to anticoagulant rodenticide resistance was found in only one brown rat specimen. Furthermore, Ser74Asn, Gln77Pro (black rat) and Ser79Pro (brown rat) mutations that cause amino acid changes in the Exon 2 region but unclear whether they cause resistance were identified. In addition, "silent mutations" which do not cause amino acid changes were also defined; these mutations were Arg12Arg mutation in Exon 1 region, His68His, Ser81Ser, Ile82Ile and Leu94Leu mutations in Exon 2 region and Ile107Ile, Thr137Thr, Ala143Ala and Gln152Gln mutations in Exon 3 region. These silent mutations were found in both species except for Ser81Ser which was determined in only brown rats.

Decreased or Absent B Cells in patients with Severe Reduction in All Serum Immunoglobulin Isotypes, a registry based study

BackgroundSevere Reduction in All Serum Immunoglobulin Isotypes with Profoundly Decreased or Absent B Cells is a subgroup of inborn error of immunities (IEI). 80 to 90% carry mutations in X-chromosomal Bruton tyrosine kinase (BTK) and around 5–10% of the cases are autosomal recessive Agammaglobulinemia (ARA). Very few of Hypogammaglobulinemia with no B cells classified in other subgroups. All of these patients benefit from a genetic evaluation, especially in highly consanguine populations. Materials and Methods27 patients from 11 families with hypogamma-globinemia and no B-cells registered in Immunodeficiency Diseases Research Center registry, Isfahan University of Medical Sciences were phenotypically and genetically evaluated. ResultsThe most prevalent genetic cause was mutations in BTK. The novel mutations in BTK gene includes c.176 G>A (p. Glu89Lys); c.115T>C (p. Tyr39His); c.685-686insTTAC (p. Asn229llefs5), in ARA includes a homozygous stop mutation in Immunoglobulin Heavy Constant Mu Chain (IGHM) gene, a frameshift mutation of B-Cell Antigen Receptor Complex-Associated Protein (CD79A), a bi-allelic stop-gain mutation in Transcription Factor 3 (TCF3), and in PIK3CD also in others IEI includes :novel homozygous silent mutations in Phosphoinositide-3-Kinase Regulatory Subunit (RASGRP1), homozygote SAMD9 mutation. DiscussionWe did not observe any phenotypically significant differences in XLA patients.The main clinical manifestation of ARA cases is, recurrent infections. However, patient with IGHM deficiency had earlier onset of the disease and more severe complications. In CD79A deficient case with 5 affected members and four deaths in her family, the symptoms started very early but managed properly according to the family history. Due to the late diagnosis of PIK3CD cases, more severe complications like bronchiectasis, neurologic sequels after poliomyelitis, and SLE nephropathy has been developed. Our novel SAMD9 had quite different phenotype and the novel RASPGR2 case with no B cells unfortunately managed as ARA before genetic diagnosis and has died of lymphoma as her 3 siblings. However, we observed that in families with more than one affected family member, the disease was better controlled in the second patient. ConclusionOur study broadens the genetic spectrum of hypogammaglobulinemia with no B cells in Iran. This helps differentiate phenotypes and genotypes and manage patients appropriately.

Single-Nucleotide Polymorphisms Identified within Exon 2 of Fertility-Associated Bone Morphogenetic Protein (BMP15) Gene in Three Romanian Sheep Breeds

The improvement of the reproductive traits of animals is of great interest for livestock production. Due to its positive impact on the sheep industry’s profitability, prolificacy is one of the most economically significant biological traits, showing variation between and within breeds of domestic sheep (Ovis aries). Different mutations in BMPR-1B, BMP15 and GDF9 genes coding for the transforming growth factor-β (TGFβ) superfamily have been shown to influence the ovulation rate and litter size. Numerous single-nucleotide polymorphisms (SNPs) in the bone morphogenetic protein 15 (BMP15) gene have been linked to ewe fecundity. Using targeted PCR amplification and Sanger sequencing, we were able to identify heterozygous SNPs in exon 2 of BMP15 in three sheep breeds reared in Romania: Tsigai, Cluj Merino and Tsurcana. The sequence analysis revealed three previously documented mutations, namely the missense mutation c.755T>C (L252P), which is predicted to change the tertiary structure of the BMP15 protein, and two silent mutations, c.747T>C (P249P) and c.1047G>A (V349V). In addition, we also identified one novel silent mutation, c.825G>A (S275S). Based on our findings and publicly available data, we indicate four putative mutational hotspots within exon 2 of BMP15 that could be considered for improving the indigenous sheep breeds through targeted gene editing and SNP genotyping strategies.

Neuropilin-1 Knockout and Rescue Confirms Its Role to Promote Metastasis in MDA-MB-231 Breast Cancer Cells.

Breast cancer (BC) metastasis remains a leading cause of female mortality. Neuropilin-1 (NRP-1) is a glycoprotein receptor that plays ligand-dependent roles in BC. Clinical studies indicate its correlation with metastatic disease; however, its functional role in BC metastasis remains uncertain. CRISPR-Cas9 was used to knockout the NRP-1 gene in MDA-MB-231 BC cells, and the effects on metastasis were determined using an orthotopic mouse engraftment model. NRP-1 expression in knockout cells was rescued using a recombinant cDNA with a silent mutation in the sgRNA target-adjacent PAM sequence. Differentially expressed genes between NRP-1 knockout and control cells were determined using whole-transcriptome sequencing and validated using real-time PCR. NRP-1KO cells showed a pronounced reduction in the metastasis to the lungs. KEGG pathway analysis of the transcriptome data revealed that PI3K and ECM receptor interactions were among the top altered pathways in the NRP-1KO cells. In addition, reduction in metastasis enhancers proteins, Integrin-β3 and Tenascin-C, and genes CCL20 and FN1 and upregulation of metastasis suppressor genes, ACVRL and GPX3 in NRP-1KO were detected. These findings provide evidence for a functional role for NRP-1 in BC metastasis, supporting further exploration of NRP-1 and the identified genes as targets in treating metastatic BC.

Phage resistance of Salmonella enterica obtained by transposon Tn5‐mediated SefR gene silent mutation

Salmonella enterica contamination is a primary cause of global food poisoning. Using phages as bactericidal alternatives to antibiotics could confront the issue of drug resistance. However, the problem of phage resistance, especially mutant strains with multiple phage resistance, is a critical barrier to the practical application of phages. In this study, a library of EZ-Tn5 transposable mutants of susceptible host S.enterica B3-6 was constructed. After the infestation pressure of a broad-spectrum phage TP1, a mutant strain with resistance to eight phages was obtained. Analysis of the genome resequencing results revealed that the SefR gene was disrupted in the mutant strain. The mutant strain displayed a reduced adsorption rate of 42% and a significant decrease in swimming and swarming motility, as well as a significantly reduced expression of the flagellar-related FliL and FliO genes to 17% and 36%, respectively. An uninterrupted form of the SefR gene was cloned into vector pET-21a (+) and used for complementation of the mutant strain. The complemented mutant exhibited similar adsorption and motility as the wild-type control. These results suggest that the disrupted flagellar-mediated SefR gene causes an adsorption inhibition, which is responsible for the phage-resistant phenotype of the S.enterica transposition mutant.

Genetic Analysis of The Leptin Gene in Goats Based on GenBank DNA Sequences

The Leptin gene is the gene that produces the leptin hormone, which is released from adipose tissue and can increase the productivity of animals. This study aimed to identify polymorphic nucleotides, changes in amino acid components, and species of goats based on GenBank Leptin DNA sequence data. A total of five goat leptin DNA sequences were extracted from NCBI GenBank data. The leptin DNA sequence was aligned with Bioedit to locate SNPs and amino acid changes. The tree produces cultivars grown using Clustal Omega Ver. 1.2.4. Based on the DNA sequencing results of leptin genes in five goats, five SNPs were located in the coding sequence (CDS), SNPs g.17T/A, g.43T/A, g.74G/A, g.93C/A. and d. 386A/G. SNP was a missense mutation and a silent mutation. The analysis of phylogenetic trees of Leptin showed that there were three breeds of goats in one branch and two breeds of goats in different branches. These results provided the first report for further studies on the genetic diversity of leptin genes in different local goat breeds.

Implication of Dynamin-2 (DNM2) Mutations in Adult T-cell Acute Lymphoblastic Leukemia.

The objective of the present study was to improve the risk stratification of T-cell Acute Lymphoblastic Leukemia (T-ALL) patients. It aimed to identify the frequency and clinical impact of DNM2 gene mutations among adult T-ALL cases. The current study included 25 T-ALL patients before starting their treatment. Mutational analysis of DNM2 gene (exons 18 and 22) was performed for all patients using Macrogen 3730 apparatus. We identified DNM2 gene mutations in 19 out of 25 (76%) patients. The detected mutations were either missense or deletion. Only active mutations (deletion) were associated with poor induction remission response and high frequency of relapse. Two novel mutations were addressed among the studied cohort of patients. They included c.1866G>C (p.V596L) and c.1872delA in exon 18. A high frequency of silent mutations was also found in T-ALL patients, but with no impact on clinical features. The DNM2 mutations were prevalent among adult T-ALL patients and might have a role in the pathogenesis of the disease. Active DNM2 mutations were associated with poor clinical outcome. Moreover, high frequency of DNM2 mutations indicated that these mutations could be utilized in detection of minimal residual disease in T-ALL patients.

Targeted Chromosomal Barcoding Establishes Direct Genotype-Phenotype Associations for Antibiotic Resistance in Mycobacterium abscessus.

A bedaquiline-resistant Mycobacterium abscessus isolate was sequenced, and a candidate mutation in the atpE gene was identified as responsible for the antibiotic resistance phenotype. To establish a direct genotype-phenotype relationship of this mutation which results in a Asp-to-Ala change at position 29 (D29A), we developed a recombineering-based method consisting of the specific replacement of the desired mutation in the bacterial chromosome. As surrogate bacteria, we used two M. abscessus bedaquiline-susceptible strains: ATCC 19977 and the SL541 clinical isolate. The allelic exchange substrates used in recombineering carried either the sole D29A mutation or a genetic barcode of silent mutations in codons flanking the D29A mutation. After selection of bedaquiline-resistant M. abscessus colonies transformed with both substrates, we obtained equivalent numbers of recombinants. These resistant colonies were analyzed by allele-specific PCR and Sanger sequencing, and we demonstrated that the presence of the genetic barcode was linked to the targeted incorporation of the desired mutation in its chromosomal location. All recombinants displayed the same MIC to bedaquiline as the original isolate, from which the D29A mutation was identified. Finally, to demonstrate the broad applicability of this method, we confirmed the association of bedaquiline resistance with the atpE A64P mutation in analysis performed in independent M. abscessus strains and by independent researchers. IMPORTANCE Antimicrobial resistance (AMR) threatens the effective prevention and treatment of an ever-increasing range of infections caused by microorganisms. On the other hand, infections caused by Mycobacterium abscessus affect people with chronic lung diseases, and their incidence has grown alarmingly in recent years. Further, these bacteria are known to easily develop AMR to the few therapeutic options available, making their treatment long-lasting and challenging. The recent introduction of new antibiotics against M. abscessus, such as bedaquiline, makes us anticipate a future when a plethora of antibiotic-resistant strains will be isolated and sequenced. However, in the era of whole-genome sequencing, one of the challenges is to unequivocally assign a biological function to each identified polymorphism. Thus, in this study, we developed a fast, robust, and reliable method to assign genotype-phenotype associations for putative antibiotic-resistant polymorphisms in M. abscessus.

Concurrent Assessment of Oxidative Stress and MT-ATP6 Gene Profiling to Facilitate Diagnosis of Autism Spectrum Disorder (ASD) in Tamil Nadu Population.

Autism spectrum disorder (ASD) is a neurodevelopmental disability that causes social impairment, debilitated verbal or nonverbal conversation, and restricted/repeated behavior. Recent research reveals that mitochondrial dysfunction and oxidative stress might play a pivotal role in ASD condition. The goal of this case-control study was to investigate oxidative stress and related alterations in ASD patients. In addition, the impact of mitochondrial DNA (mtDNA) mutations, particularly MT-ATP6, and its link with oxidative stress in ASD was studied. We found that ASD patient's plasma had lower superoxide dismutase (SOD) and higher catalase (CAT) activity, resulting in lower SOD/CAT ratio. MT-ATP6 mutation analysis revealed that four variations, 8865 G>A, 8684 C>T, 8697 G>A, and 8836 A>G, have a frequency of more than 10% with missense and synonymous (silent) mutations. It was observed that abnormalities in mitochondrial complexes (I, III, V) are more common in ASD, and it may have resulted in MT-ATP6 changes or vice versa. In conclusion, our findings authenticate that oxidative stress and genetics both have an equal and potential role behind ASD and we recommend to conduct more such concurrent research to understand their unique mechanism for better diagnosis and therapeutic for ASD.

Blue-light background impairs visual exogenous attention shift

Previous research into the effects of blue light on visual-spatial attention has yielded mixed results due to a lack of properly controlling critical factors like S-cone stimulation, ipRGCs stimulation, and color. We adopted the clock paradigm and systematically manipulated these factors to see how blue light impacts the speed of exogenous and endogenous attention shifts. Experiments 1 and 2 revealed that, relative to the control light, exposure to the blue-light background decreased the speed of exogenous (but not endogenous) attention shift to external stimuli. To further clarify the contribution(s) of blue-light sensitive photoreceptors (i.e., S-cone and ipRGCs), we used a multi-primary system that could manipulate the stimulation of a single type of photoreceptor without changing the stimulation of other photoreceptors (i.e., the silent substitution method). Experiments 3 and 4 revealed that stimulation of S-cones and ipRGCs did not contribute to the impairment of exogenous attention shift. Our findings suggest that associations with blue colors, such as the concept of blue light hazard, cause exogenous attention shift impairment. Some of the previously documented blue-light effects on cognitive performances need to be reevaluated and reconsidered in light of our findings.

Abstract P2-23-13: Highly proliferative estrogen receptor-positive breast cancer is associated with better response to neoadjuvant chemotherapy but worse clinical outcome

Abstract Introduction: Highly proliferative cancers are known to respond better to neoadjuvant chemotherapy (NAC), and pathological complete response (pCR) is a surrogate for survival benefit in breast cancer. Ki67 is the most commonly used cell proliferation marker; however, conventional immunostaining is subjected to variability by the assessor and the institution including the antibody used. To this end, we used the Ki67 gene expression to identify highly proliferative breast cancer and investigated whether it is associated with better response to NAC and survival by intrinsic subtype. Materials and Methods: Total of 6623 breast cancer patients from 11 independent cohorts with full transcriptome and clinical data were analyzed. The top 20% of Ki67 gene expression in each cohort were defined as highly proliferative group based on previous studies using immuno-staining. Results: Highly proliferative breast cancer was associated with high proliferation score, and all five cell proliferation-related gene sets in the Hallmark collection; E2F Targets, G2M Checkpoint, Mitotic Spindle, and Myc Targets v1 and v2 were enriched by gene set enrichment analysis in multiple cohorts regardless of subtype. MKI67 gene expression increased with higher grade in both ER-positive breast cancer and TNBC in multiple cohorts, but no clear association with staging was observed. Highly proliferative ER-positive breast cancer was significantly associated with worse disease-free, disease-specific, and overall survival consistently in METABRIC and GSE96058 cohorts, and showed a trend toward worse overall survival in TCGA; however, this was not the case in TNBC. Further, we found that highly proliferative tumor was significantly associated with better pathologic complete response (pCR) rate in six of the eight neoadjuvant chemotherapy cohorts of ER-positive breast cancer, but only two for TNBC. We also found that highly proliferative ER-positive breast cancer was associated with abundant silent and non-silent mutations, increased intra-tumoral heterogeneity, and homologous recombination defect scores when compared to lower proliferation groups, and this was not seen in TNBC in TCGA cohort. Although DNA repair gene set was enriched in the highly proliferative ER-positive breast cancer, it was also associated with higher neoantigen activity, higher infiltration of tumor infiltrating lymphocytes including Th1 cells, Th2 cells, regulatory T cells, and M1 macrophages, as well as with enrichment of Interferon-gamma response gene set consistently in 3 large cohorts. In contrast, several stromal cell fractions were decreased in this group. Conclusions: Highly proliferative ER-positive breast cancer is significantly associated with pCR after NAC with increased tumor infiltrating lymphocytes, but is not a surrogate for survival, which are not the case in TNBC. Citation Format: Rongrong Wu, Yoshihisa Tokumaru, Mariko Asaoka, Masanori Oshi, Li yan, Takashi Ishikawa, Kazuaki Takabe. Highly proliferative estrogen receptor-positive breast cancer is associated with better response to neoadjuvant chemotherapy but worse clinical outcome [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-23-13.

Abstract PD2-04: Preclinical development of CodaLytic™, a codon-modified influenza virus, as a novel virotherapeutic agent for breast cancer immunotherapy

Abstract Multiple species of oncolytic viruses (OVs) have been shown to modulate the tumor microenvironment (TME) by increasing immune cell infiltration and activating stimulatory immune responses, leading to the induction of a tumor-specific immune response. Many engineered OVs achieve tumor specificity by either gene deletion or mutations and are then armed with immunomodulatory transgenes to promote anti-tumor immune responses. In an alternative approach, OVs derived from Codagenix’s codon/codon pair modification platform aim to leverage the natural immunostimulatory capacity of selected viral species for efficacy and take advantage of defects in innate immune sensing and apoptosis mechanisms in cancer cells as well as receptor overexpression for tumor selectivity. CodaLytic is a novel virotherapeutic derived from influenza virus strain A/California/07/2009, that is synthetically engineered to contain over 600 silent mutations in hemagglutinin and neuraminidase genes and is being developed as a novel immunotherapeutic for breast cancer. In the orthotopic EMT6 triple-negative breast cancer model, known for its moderate sensitivity to immunotherapies, intratumoral injection of 108 PFU three times a week for up to 4 weeks as a monotherapy led to significant tumor growth inhibition by 76% (p < 0.001 vs vehicle control), translating into a significant survival benefit with a 66% cure rate. Intravenous rechallenge of EMT6 long-term survivors led to a 27-fold reduction in lung nodule formation and a tumor-specific interferon-γ memory response was observed ex vivo in their splenocytes. Anti-tumor efficacy after CodaLytic treatment was accompanied by a change in the composition of the tumor immune infiltrate with significant increases in T, B and NK cells and increased gene expression of pathways and genes related to T cell effector function, dendritic cell activation, antigen presentation and chemoattraction. In immunotherapy-resistant orthotopic 4T1 tumors, combination of CodaLytic with a CTLA-4 inhibitory antibody, but not anti-PD-1 blockade – a combination that had demonstrated combination benefit in several other preclinical models – led to reduction in tumor growth by 75% (p < 0.0001 vs vehicle control and PD-1 combination group). Median overall survival improved from 22 to 30 days with addition of CTLA-4 blockade. Triple combination including PD-1 inhibition led to improved long-term survival beyond 50 days with 30% complete regressions. In summary, these preclinical data demonstrate CodaLytic’s ability to induce broad innate and adaptive changes in the breast cancer TME, resulting in anti-tumor efficacy and prolonged survival. Characterization of the tumor immune infiltrate after CTLA-4 combination and pharmacodynamic changes achieved after CodaLytic/checkpoint combination treatment in human breast cancer tumoroids will further support identification of correlates of efficacy with translational implications. Together with preclinical toxicology data and demonstrated clinical safety of this attenuated influenza virus after intranasal administration in healthy individuals, CodaLytic emerges as a promising novel viroimmunotherapeutic agent and is planned to enter a phase 1 clinical trial in early 2023. Citation Format: Marcin Stawowczyk, Yiwen Zhao, Katie Pfeffer, Juliana Tafrova, James Rodriguez, Chen Yang, Nusrat Jahan, Sybil A. Tasker, Steffen Mueller, J. Robert Coleman, Johanna K. Kaufmann. Preclinical development of CodaLytic™, a codon-modified influenza virus, as a novel virotherapeutic agent for breast cancer immunotherapy [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD2-04.

Microscopical and phylogenetic analysis of Theileria annulata in Iraqi local breed cattle

The development of cattle industry in Iraq and its neighboring countries may be hampered by Theileria annulata (T. annulata) infection. In the present work, the cytochrome b gene (cyt-b) sequences were used to infer the phylogenetic relationships of T. annulata. Fifty blood samples collected randomly from 10 farms of Iraqi local breed cattle in Al-Diwaniyah province were analyzed for piroplasm using microscopic and molecular techniques. Microscopic examination revealed that 37 (74%) of the 50 blood samples examined were infected. In addition, the PCR test showed that 23 (62.2%) of the 37 blood samples (which tested positive (microscopically) were positive. The analysis of the cyt-b 1092 bp gene of T. annulata reported the presence of six genetically related clones. These six clones have been deposited in GenBank as [ON706262, ON706263, ON706264, ON706265, ON706266, and ON706267], and when compared to the T. annulata cyt-b gene (XM949625) from the GenBank database, a unique polymorphism at sixteen sites was discovered. Furthermore, the alignment of our amino acid sequences with the reference sequence reveals thirteen non-synonymous mutations in codons 11, 22, 33, 63, 103, 128, 130, 129, 172,178, and 190, and three silent mutations in codons 124, 128, and 275. Moreover, phylogenetically, our study clones were related to Iraqi, Indian, and Turkish clones. In conclusion, depending on the phylogenetic analysis the current study identified six T. annulata parasite isolates in Iraqi local breed cattle that were genetically related to the Indian, Iraqi, and Turkish clones.

Genetic variability of the prion protein gene in Indonesian goat breeds

Scrapie is a naturally occurring transmissible spongiform encephalopathy in sheep and goats. Resistance or susceptibility of small ruminants to classical scrapie is influenced by polymorphisms in the prion protein gene (PRNP). PRNP variability in Indonesian indigenous goat breeds has not been investigated so far and therefore was the goal of this study. Sanger sequencing of the PRNP gene coding region in 72 goats of the seven Indonesian breeds Kacang, Gembrong, Samosir, Kejobong, Benggala, Jawarandu, and Peranakan Etawah revealed three amino acid substitutions, namely W102G, H143R, and S240P. Some silent mutations were also found at codons 42 (a/g), 138 (c/t), and 179 (g/t). The PRNP alleles K222 and D/S146 known to have significant protective effects on resistance to classical scrapie in goats were not detected. The allele R143, which may have a moderate protective effect, had a frequency of 12% among the analyzed Indonesian goat breeds. While R143 was missing in Kacang and Benggala, its frequency was highest in the breed Gembrong (32%). No scrapie cases have been reported in Indonesia until now. However, in the case that selection for protective PRNP variants would become a breeding goal, the analyzed breeds will not be very useful resources. Other goat breeds which are present in the country should be investigated regarding resistance to scrapie, too.

Single nucleotide polymorphisms within exon four of the prolactin gene and their effect on milk traits in cattle populations of Ethiopia

Bovine prolactin (PRL) gene is essential for the initiation and maintenance of lactation and exerts multiple effects on mammary alveoli to promote the synthesis and secretion of major components of milk. The objectives of this study were to identify mutations in PRL gene and to evaluate the mutations as potential markers of milk performance traits in cattle populations of Ethiopia. For this purpose, genomic DNA from whole blood was extracted through salting out procedure from 87 animals of five cattle populations of Ethiopia. Accordingly, three single nucleotide polymorphisms (SNPs) were identified of which one SNP g.8323T > A showed missense mutation while the other two SNPs revealed silent mutations. FST values showed statistically significant genetic differentiation among the studied populations. Intermediate polymorphic information content was noted for most SNPs, which indicates the presence of sufficient genetic variation at this locus. Two SNPs showed heterozygote deficiency as a result of positive FIS values. Only g.8398A > G SNP have statistically significant (p < 0.05) effect on average daily milk yield, fat and solid not fat percentage in all studied cattle populations. Therefore, g.8398A > G SNP identified in this study influences cattle milk production and may be used as possible candidate SNP for marker-assisted selection programs in cattle populations of Ethiopia.