Haploid androgenetic development of bovine embryos reveals imbalanced WNT signaling and impaired cell fate differentiation†.

Haploid embryos have contributed significantly to our understanding of the role of parental genomes in development and can be applied to important biotechnology for human and animal species. However, development to the blastocyst stage is severely hindered in bovine haploid androgenetic embryos (hAE). To further our understanding of such developmental arrest, we performed a comprehensive comparison of the transcriptomic profile of morula-stage embryos, which were validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) of transcripts associated with differentiation in haploid and biparental embryos. Among numerous disturbances, results showed that pluripotency pathways, especially the wingless-related integration site (WNT) signaling, were particularly unbalanced in hAE. Moreover, transcript levels of KLF4, NANOG, POU5F1, SOX2, CDX2, CTNNBL1, AXIN2, and GSK3B were noticeably altered in hAE, suggesting disturbance of pluripotency and canonical WNT pathways. To evaluate the role of WNT on hAE competence, we exposed early Day-5 morula stage embryos to the GSK3B inhibitor CHIR99021. Although no alterations were observed in pluripotency and WNT-related transcripts, exposure to CHIR99021 improved their ability to reach the blastocysts stage, confirming the importance of the WNT pathway in the developmental outcome of bovine hAE.

Characterization of histone lysine ß-hydroxybutyrylation in bovine tissues, cells, and cumulus-oocyte complexes.

Besides their canonical roles as energy sources, short-chain fatty acids act as metabolic regulators of gene expression through histone posttranslational modifications. Ketone body ß-hydroxybutyrate (BHB) causes a novel epigenetic modification, histone lysine ß-hydroxybutyrylation (Kbhb), which is associated with genes upregulated in starvation-responsive metabolic pathways. Dairy cows increase BHB in early lactation, and the effects of this increase on cellular epigenomes are unknown. We searched for and identified that Kbhb is present in bovine tissues in vivo and confirmed that this epigenetic mark is responsive to BHB in bovine and human fibroblasts cultured in vitro in a dose-dependent manner. Maturation of cumulus-oocyte complexes with high concentrations of BHB did not affect the competence to complete meiotic maturation or to develop until the blastocyst stage. BHB treatment strongly induced H3K9bhb in cumulus cells, but faintly in oocytes. RNA-seq analysis in cumulus cells indicated that BHB treatment altered the expression of 345 genes. The downregulated genes were mainly involved in glycolysis and ribosome assembly pathways, while the upregulated genes were involved in mitochondrial metabolism and oocyte development. The genes and pathways altered by BHB will provide entry points to carry out functional experiments aiming to mitigate metabolic disorders and improve fertility in cattle.

Resiliency of equid H19 imprint to somatic cell reprogramming by oocyte nuclear transfer and genetically induced pluripotency†.

Cell reprogramming by somatic cell nuclear transfer and in induced pluripotent stem cells is associated with epigenetic modifications that are often incompatible with embryonic development and differentiation. For instance, aberrant DNA methylation patterns of the differentially methylated region and biallelic expression of H19-/IGF2-imprinted gene locus have been associated with abnormal growth of fetuses and placenta in several mammalian species. However, cloned horses are born with normal sizes and with no apparent placental anomalies, suggesting that H19/IGF2 imprinting may be epigenetically stable after reprogramming in this species. In light of this, we aimed at characterizing the equid H19 gene to determine whether imprinting is altered in somatic cell nuclear transfer (SCNT)-derived conceptuses and induced pluripotent stem cell (iPSC) lines using the mule hybrid model. A CpG-rich region containing five CTCF binding sites was identified upstream of the equine H19 gene and analyzed by bisulfite sequencing. Coupled with parent-specific and global H19 transcript analysis, we found that the imprinted H19 remains monoallelic and that on average the methylation levels of both parental differentially methylated regions in embryonic and extra-embryonic SCNT tissues and iPSC lines remained unaltered after reprogramming. Together, these results show that, compared to other species, equid somatic cells are more resilient to epigenetic alterations to the H19-imprinted locus during SCNT and iPSC reprogramming.

The Infertility of Repeat-Breeder Cows During Summer Is Associated with Decreased Mitochondrial DNA and Increased Expression of Mitochondrial and Apoptotic Genes in Oocytes.

Oocyte quality is known to be a major cause of infertility in repeat-breeder (RB) and heat-stressed dairy cows. However, the mechanisms by which RB oocytes become less capable of supporting embryo development remain largely unknown. Thus, the aim of this study was to investigate whether the decreased oocyte competence of RB cows (RBs) during summer is associated with an altered gene expression profile and a decrease in mitochondrial DNA (mtDNA) copy number. Therefore, oocytes collected from heifers, non-RBs in peak lactation (PLs), and RBs were used to evaluate mtDNA amounts as well as the expression levels of genes associated with the mitochondria (MT-CO1, NRF1, POLG, POLG2, PPARGC1A, and TFAM), apoptosis (BAX, BCL2, and ITM2B), and oocyte maturation (BMP15, FGF8, FGF10, FGF16, FGF17, and GDF9). The oocytes retrieved from RBs during winter contained over eight times more mtDNA than those retrieved from RBs during summer. They also contained significantly less mtDNA than oocytes retrieved from heifers and PLs during summer. Moreover, the expression of mitochondria- (NRF1, POLG, POLG2, PPARGC1A, and TFAM) and apoptosis-related (BAX and ITM2B) genes, as well as of GDF9, in RB oocytes collected during summer was significantly greater than that in oocytes collected from heifers and PLs during the same season. In oocytes from heifers and PLs, the expression levels of these genes were lower in those collected during summer compared with winter, but this difference was not observed in oocytes collected from RBs. Altogether, these data provide evidence of altered gene expression and reduced mtDNA copy number in the oocytes collected from RBs during summer. This indicates a loss of fertility in RBs during summer, which might be caused by a possible mitochondrial dysfunction associated with a greater chance of oocytes to undergo apoptosis.

Messenger RNAs in metaphase II oocytes correlate with successful embryo development to the blastocyst stage.

The mRNAs accumulated in oocytes provide support for embryo development until embryo genomic activation. We hypothesized that the maternal mRNA stock present in bovine oocytes is associated with embryo development until the blastocyst stage. To test our hypothesis, we analyzed the transcriptome of the oocyte and correlated the results with the embryo development. Our goal was to identify genes expressed in the oocyte that correlate with its ability to develop to the blastocyst stage. A fraction of oocyte cytoplasm was biopsied using micro-aspiration and stored for further expression analysis. Oocytes were activated chemically, cultured individually and classified according to their capacity to develop in vitro to the blastocyst stage. Microarray analysis was performed on mRNA extracted from the oocyte cytoplasm fractions and correlated with its ability to develop to the blastocyst stage (good quality oocyte) or arrest at the 8-16-cell stage (bad quality oocyte). The expression of 4320 annotated genes was detected in the fractions of cytoplasm that had been collected from oocytes matured in vitro. Gene ontology classification revealed that enriched gene expression of genes was associated with certain biological processes: 'RNA processing', 'translation' and 'mRNA metabolic process'. Genes that are important to the molecular functions of 'RNA binding' and 'translation factor activity, RNA binding' were also enriched in oocytes. We identified 29 genes with differential expression between the two groups of oocytes compared (good versus bad quality). The content of mRNAs expressed in metaphase II oocytes influences the activation of the embryonic genome and enables further develop to the blastocyst stage.

Digital Multiphase Composites via Additive Manufacturing.

Mechanical properties of traditional engineering materials are typically coupled to each other, presenting a challenge to practitioners with multi-dimensional material property requirements. In this work, continuous, independent control over multiple mechanical properties is demonstrated in composite materials realized using additive manufacturing. For the first time, composites additively manufactured from rigid plastic, soft elastomer, and liquid constituents are experimentally characterized, demonstrating materials which span four orders of magnitude in modulus and two orders of magnitude in toughness. By forming analytical mappings between relative concentrations of constituents at the microscale and resulting macroscale material properties, inverse material design is enabled; the method is showcased by printing artifacts with prescribed toughness and elasticity distributions. The properties of these composites are placed in the context of biological tissues, showing they have promise as mechanically plausible tissue mimics.

Rapid dynamic changes of FL.2 variant: A case report of COVID-19 breakthrough infection.

We investigated intra-host genetic evolution using two SARS-CoV-2 isolates from a fully vaccinated (primary schedule x2 doses of AstraZeneca plus a booster of Pfizer), >70-year-old woman with a history of lymphoma and hypertension who presented a SARS-CoV-2 infection for 3 weeks prior to death due to COVID-19. Two full genome sequences were determined from samples taken 13 days apart with both belonging to Pango lineage FL.2: the first detection of this Omicron sub-variant in Botswana. FL.2 is a sub-lineage of XBB.1.9.1. The repertoire of mutations and minority variants in the Spike protein differed between the two time points. Notably, we also observed deletions within the ORF1a and Membrane proteins; both regions are associated with high T-cell epitope density. The internal milieu of immune-suppressed individuals may accelerate SARS-CoV-2 evolution; hence, close monitoring is warranted.

Comparison of the Multiple Mini-Interview and the Traditional Interview in Medical School Admissions: Lessons Learned Using a Hybrid Model at One Institution.

PURPOSE: Medical school admissions interviews are a critical form of assessment; however, the most effective interview strategy is debated. This study compares the traditional interview (TI) and multiple mini-interview (MMI) within a hybrid TI-MMI model at one medical school to determine whether the interview approaches reveal different information about applicants and whether a hybrid model results in a more diversified applicant pool. METHOD: Admissions data from 3 application cycles at the Donald and Barbara Zucker School of Medicine at Hofstra/Northwell were used. The TI was used in 2017-2018 and the hybrid TI-MMI model in 2018-2019 and 2019-2020. Applicants were scored on a 5-point scale and referred to a voting committee for acceptance consideration if interview scores met threshold criteria. Changes in the number of students referred to the committee using the TI vs the TI-MMI score criteria were analyzed. RESULTS: In 2017-2018 (TI only), 683 applicants were interviewed; in 2018-2019 (TI-MMI), 844 applicants were interviewed; and in 2019-2020 (TI-MMI), 805 applicants were interviewed. Medium correlations were found between total MMI and TI scores in 2018-2019 ( ρ = 0.37, P < .001) and 2019-2020 ( ρ = 0.33, P < .001). No differences were found in TI scores between 2017-2018 and 2018-2019 ( P = .30), but TI scores were significantly lower in 2019-2020 vs 2017-2018 ( P < .001) and 2018-2019 ( P = .002). Overall, a 10% to 18% increase was found in the number of applicants referred to the voting committee when using hybrid criteria, with a 19% to 27% increase in underrepresented in medicine applicants. CONCLUSIONS: The TI-MMI model may allow for a more holistic interview approach and an expanded pool of applicants, particularly underrepresented in medicine applicants, considered for acceptance.

Cattle Cloning by Somatic Cell Nuclear Transfer.

Cloning by somatic cell Nuclear Transfer (SCNT) is a powerful technology capable of reprograming terminally differentiated cells to totipotency for generating whole animals or pluripotent stem cells for use in cell therapy, drug screening, and other biotechnological applications. However, the broad usage of SCNT remains limited due to its high cost and low efficiency in obtaining live and healthy offspring. In this chapter, we first briefly discuss the epigenetic constraints responsible for the low efficiency of SCNT and current attempts to overcome them. We then describe our bovine SCNT protocol for delivering live cloned calves and addressing basic questions about nuclear reprogramming. Other research groups can benefit from our basic protocol and build up on it to improve SCNT in the future. Strategies to correct or mitigate epigenetic errors (e.g., correcting imprinting loci, overexpression of demethylases, chromatin-modifying drugs) can integrate the protocol described here.

The Inner Complexities of Multimodal Medical Data: Bitmap-Based 3D Printing for Surgical Planning Using Dynamic Physiology.

Motivated by the need to develop more informative and data-rich patient-specific presurgical planning models, we present a high-resolution method that enables the tangible replication of multimodal medical data. By leveraging voxel-level control of multimaterial three-dimensional (3D) printing, our method allows for the digital integration of disparate medical data types, such as functional magnetic resonance imaging, tractography, and four-dimensional flow, overlaid upon traditional magnetic resonance imaging and computed tomography data. While permitting the explicit translation of multimodal medical data into physical objects, this approach also bypasses the need to process data into mesh-based boundary representations, alleviating the potential loss and remodeling of information. After evaluating the optical characteristics of test specimens generated with our correlative data-driven method, we culminate with multimodal real-world 3D-printed examples, thus highlighting current and potential applications for improved surgical planning, communication, and clinical decision-making through this approach.

Placental scaffolds as a potential biological platform for embryonic stem cells differentiation into hepatic-like cells lineage: A pilot study.

Hepatic microenvironment plays an essential role in liver regeneration, providing the necessary conditions for cell proliferation, differentiation and tissue rearrangement. One of the key factors for hepatic tissue reconstruction is the extracellular matrix (ECM), which through collagenous and non-collagenous proteins provide a three-dimensional structure that confers support for cell adhesion and assists on their survival and maintenance. In this scenario, placental ECM may be eligible for hepatic tissue reconstruction, once these scaffolds hold the major components required for cell support. Therefore, this preliminary study aimed to access the possibility of mouse embryonic stem cells differentiation into hepatocyte-like cells on placental scaffolds in a three-dimensional dynamic system using a Rotary Cell Culture System. Following a four-phase differentiation protocol that simulates liver embryonic development events, the preliminary results showed that a significant quantity of cells adhered and interacted with the scaffold through outer and inner surfaces. Positive immunolabelling for alpha fetus protein and CK7 suggest presence of hepatoblast phenotype cells, and CK18 and Albumin positive immunolabelling suggest the presence of hepatocyte-like phenotype cells, demonstrating the presence of a heterogeneous population into the recellularized scaffolds. Periodic Acid Schiff-Diastase staining confirmed the presence of glycogen storage, indicating that differentiate cells acquired a hepatic-like phenotype. In conclusion, these preliminary results suggested that mouse placental scaffolds might be used as a biological platform for stem cells differentiation into hepatic-like cells and their establishment, which may be a promissing biomaterial for hepatic tissue reconstruction.

Exogenous OCT4 and SOX2 Contribution to In Vitro Reprogramming in Cattle.

Mechanisms of cell reprogramming by pluripotency-related transcription factors or nuclear transfer seem to be mediated by similar pathways, and the study of the contribution of OCT4 and SOX2 in both processes may help elucidate the mechanisms responsible for pluripotency. Bovine fibroblasts expressing exogenous OCT4 or SOX2, or both, were analyzed regarding the expression of pluripotency factors and imprinted genes H19 and IGF2R, and used for in vitro reprogramming. The expression of the H19 gene was increased in the control sorted group, and putative iPSC-like cells were obtained when cells were not submitted to cell sorting. When sorted cells expressing OCT4, SOX2, or none (control) were used as donor cells for somatic cell nuclear transfer, fusion rates were 60.0% vs. 64.95% and 70.53% vs. 67.24% for SOX2 vs. control and OCT4 vs. control groups, respectively; cleavage rates were 66.66% vs. 81.68% and 86.47% vs. 85.18%, respectively; blastocyst rates were 33.05% vs. 44.15% and 52.06% vs. 44.78%, respectively. These results show that the production of embryos by NT resulted in similar rates of in vitro developmental competence compared to control cells regardless of different profiles of pluripotency-related gene expression presented by donor cells; however, induced reprogramming was compromised after cell sorting.

Near-complete genome of SARS-CoV-2 Delta variant of concern identified in a symptomatic dog (Canis lupus familiaris) in Botswana.

We sought to investigate whether SARS-CoV-2 was present, and to perform full-length genomic sequencing, in a 5-year-old male crossbreed dog from Gaborone, Botswana that presented overt clinical signs (flu-like symptoms, dry hacking cough and mild dyspnoea). It was only sampled a posteriori, because three adult owners were diagnosed with SARS-CoV-2 infection. Next-generation sequencing based on Oxford Nanopore Technology (ONT) was performed on amplicons that were generated using a reverse transcriptase real-time polymerase chain reaction (RT-qPCR) of confirmed positive SARS-CoV-2 nasopharyngeal and buccal swabs, as well as a bronchoalveolar lavage with mean real cycle threshold (qCt) value of 36 based on the Nucleocapsid (N) gene. Descriptive comparisons to known sequences in Botswana and internationally were made using mutation profiling analysis and phylogenetic inferences. Human samples were not available. A near-full length SARS-CoV-2 genome (∼90% coverage) was successfully genotyped and classified under clade 20 O and Pango-Lineage AY.43 (Pango v.4.0.6 PLEARN-v1.3; 2022-04-21), which is a sublineage of the Delta variant of concern (VOC) (formerly called B.1.617.2, first detected in India). We did not identify novel mutations that may be used to distinguish SARS-CoV-2 isolates from the dog and humans. In addition to Spike (S) region mutation profiling, we performed phylogenetic analysis including 30 Delta sequences publicly available reference also isolated from dogs. In addition, we performed another exploratory analysis to investigate the phylogenetic relatedness of sequence isolated from dog with those from humans in Botswana (n = 1303) as of 31 March 2022 and of same sublineage. Expectedly, the sequence formed a cluster with Delta sublineages - AY.43, AY.116 and B.1.617.2 - circulating in same time frame. This is the first documented report of human-associated SARS-CoV-2 infection in a dog in Botswana. Although the direction of transmission remains unknown, this study further affirms the need for monitoring pets during different COVID-19 waves for possible clinically relevant SARS-CoV-2 transmissions between species.

Expression of steroidogenic proteins in bovine placenta during the first half of gestation.

The aim of the present study was to determine the occurrence and localisation of the principal steroidogenic proteins in bovine placenta from Day 50 to Day 120 of pregnancy. Immunohistochemistry revealed that, at all stages investigated, bovine steroidogenic acute regulatory protein (StAR), cytochrome P45011A1 and hydroxy-δ-5-steroid dehydrogenase, 3ß- and steroid δ-isomerase 1 proteins were found principally at the fetomaternal interdigitations: the chorionic villus and maternal septum. Moreover, caruncular epithelial cells and uninucleate trophoblast cells were the principal cells detected that were positive for the three markers. Western blot analysis showed that only caruncular tissue expressed all three steroidogenic markers; in contrast, cotyledons only expressed StAR and cytochrome P45011A1. Immunoblot results showed a complementary pattern of StAR and cytochrome P45011A1 expression between caruncles and cotyledons at different stages. These observations suggest that, in early pregnancy, the maternal compartment contributes significantly to bovine placental steroidogenesis, particularly for the synthesis of progesterone. Furthermore, the variation in StAR and cytochrome P45011A1 expression between caruncular and cotyledonary tissues across gestation suggests that placental steroidogenesis requires cell-to-cell communication between maternal and fetal cells.

Voxel Printing Anatomy: Design and Fabrication of Realistic, Presurgical Planning Models through Bitmap Printing.

Most applications of 3-dimensional (3D) printing for presurgical planning have been limited to bony structures and simple morphological descriptions of complex organs due to the fundamental limitations in accuracy, quality, and efficiency of the current modeling paradigm. This has largely ignored the soft tissue critical to most surgical specialties where the interior of an object matters and anatomical boundaries transition gradually. Therefore, the needs of the biomedical industry to replicate human tissue, which displays multiple scales of organization and varying material distributions, necessitate new forms of representation. Presented here is a novel technique to create 3D models directly from medical images, which are superior in spatial and contrast resolution to current 3D modeling methods and contain previously unachievable spatial fidelity and soft tissue differentiation. Also presented are empirical measurements of novel, additively manufactured composites that span the gamut of material stiffnesses seen in soft biological tissues from MRI and CT. These unique volumetric design and printing methods allow for deterministic and continuous adjustment of material stiffness and color. This capability enables an entirely new application of additive manufacturing to presurgical planning: mechanical realism. As a natural complement to existing models that provide appearance matching, these new models also allow medical professionals to "feel" the spatially varying material properties of a tissue simulant-a critical addition to a field in which tactile sensation plays a key role.

Defining Soft Tissue: Bitmap Printing of Soft Tissue for Surgical Planning.

Nearly all applications of 3D printing for surgical planning have been limited to bony structures and simple morphological descriptions of complex organs due to the fundamental limitations in accuracy, quality, and efficiency of the current modeling paradigms and technologies. Current approaches have largely ignored the constitution of soft tissue critical to most surgical specialties where multiple high-resolution variations transition gradually across the interior of the volume. Differences in the scales of organization related to unique organs require special attention to capture fine features critical to surgical procedures. We present a six-material bitmap printing technique for creating 3D models directly from medical images, which are superior in spatial and contrast resolution to current 3D modeling methods, and contain previously unachievable spatial fidelity for soft tissue differentiation. A retrospective exempt IRB was obtained for all data through the Colorado Multiple Institution Review Board #21-3128.

Rapid Fabrication of Low-Cost Thermal Bubble-Driven Micro-Pumps.

Thermal bubble-driven micro-pumps are an upcoming actuation technology that can be directly integrated into micro/mesofluidic channels to displace fluid without any moving parts. These pumps consist of high power micro-resistors, which we term thermal micro-pump (TMP) resistors, that locally boil fluid at the resistor surface in microseconds creating a vapor bubble to perform mechanical work. Conventional fabrication approaches of thermal bubble-driven micro-pumps and associated microfluidics have utilized semiconductor micro-fabrication techniques requiring expensive tooling with long turn around times on the order of weeks to months. In this study, we present a low-cost approach to rapidly fabricate and test thermal bubble-driven micro-pumps with associated microfluidics utilizing commercial substrates (indium tin oxide, ITO, and fluorine doped tin oxide, FTO, coated glass) and tooling (laser cutter). The presented fabrication approach greatly reduces the turn around time from weeks/months for conventional micro-fabrication to a matter of hours/days allowing acceleration of thermal bubble-driven micro-pump research and development (R&D) learning cycles.

COVID-19 in Princess Marina Hospital, Botswana: An Outbreak Investigation.

The Princess Marina Hospital in Gaborone, Botswana, had an outbreak of COVID-19 from early August 2020. The aim of this paper was to describe the outbreak investigation. The investigation's specific objectives were to describe the COVID-19 cases in terms of person, place, and time (PPT) and to determine measures to prevent further transmission of the infection. The data reported herein were collected over a 3-month period from beginning of August to end of October 2020. The investigation included all COVID-19 cases i.e. both patients and healthcare workers. It followed the steps of an outbreak investigation. These included assembling an investigation team comprising both the hospital and DHMT staff. All the wards reported their confirmed cases to the infection control team who in turn prepared line lists and case reports. Epicurves were produced from date of positive result. A total of 193 cases were reported, of which 110 (57.0%) were patients and 83 (43.0%) were healthcare workers. The median age was 35 years. Females accounted for 154 (79.8%) participants. Most of the wards were affected. The wards with the highest numbers of cases were female medical ward (39), emergency department (24), gynecology ward (17), and pediatric medical ward (10). Control measures included restricting movement into the hospital as well as clinical screening at all entry points. Furthermore, all patients were tested before admission into the wards. Surveillance of COVID-19 cases was continued beyond the 3 months reported in this paper. COVID-19 can spread rapidly in hospital settings affecting both patients and healthcare workers. Outbreak investigations including describing cases in terms of person, place, and time are critical if the most effective and efficient control measures are to be implemented.

Recellularized rat testis scaffolds with embryoid bodies cells: a promising approach for tissue engineering.

Tissue engineering is gaining use to investigate the application of its techniques for infertility treatment. The use of pluripotent embryonic cells for in vitro production of viable spermatozoa in testicular scaffolds is a promising strategy that could solve male infertility. Due to cell-extracellular matrix (ECM) interactions, here we aim to investigate the differentiation of embryoid bodies (EBs) in cultured into decellularized rat testis scaffolds. Decellularized testis (P = 0.019) with a low concentration of gDNA (30.58 mg/ng tissue) was obtained by sodium dodecyl sulfate perfusion. The structural proteins (collagens type I and III) and the adhesive glycoproteins of ECM (laminin and fibronectin) were preserved according to histological and scanning electron microscopy (SEM) analyses. Then, decellularized rat testis were cultured for 7 days with EB, and EB mixed with retinoic acid (RA) in non-adherent plates. By SEM, we observe that embryonic stem cells adhered in the decellularized testis ECM. By immunofluorescence, we verified the positive expression of HSD17B3, GDNF, ACRV-1, and TRIM-36, indicating their differentiation using RA in vitro, reinforcing the possibility of EB in male germ cell differentiation. Finally, recellularized testis ECM may be a promising tool for future new approaches for testicular cell differentiation applied to assisted reproduction techniques and infertility treatment.Abbreviations: ACRV-1: Acrosomal vesicle protein 1; ATB: Penicillin-streptomycin; DAPI: 4,6-Diamidino-2-phenylindole; EB: Embryoid bodies; ECM: Extracellular matrix; ESCs: Pluripotent embryonic stem cells; GAGs: Glycosaminoglycans; gDNA: Genomic DNA; GDNF: Glial cell line-derived neurotrophic factor; H&E: Hematoxylin and eosin; HSD17B3: 17-beta-Hydroxysteroid dehydrogenase type 3; PBS: Phosphate-buffered saline; PGCLCs: Primordial germ-cell-like cells; RA: Retinoic acid; SDS: Sodium dodecyl sulfate; SEM: Scanning electron microscopy; SSCs: Spermatogonial stem cells; TRIM-36: Tripartite Motif Containing 36.

Emergence of SARS-CoV-2 Omicron lineages BA.4 and BA.5 in South Africa.

Three lineages (BA.1, BA.2 and BA.3) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant of concern predominantly drove South Africa's fourth Coronavirus Disease 2019 (COVID-19) wave. We have now identified two new lineages, BA.4 and BA.5, responsible for a fifth wave of infections. The spike proteins of BA.4 and BA.5 are identical, and similar to BA.2 except for the addition of 69-70 deletion (present in the Alpha variant and the BA.1 lineage), L452R (present in the Delta variant), F486V and the wild-type amino acid at Q493. The two lineages differ only outside of the spike region. The 69-70 deletion in spike allows these lineages to be identified by the proxy marker of S-gene target failure, on the background of variants not possessing this feature. BA.4 and BA.5 have rapidly replaced BA.2, reaching more than 50% of sequenced cases in South Africa by the first week of April 2022. Using a multinomial logistic regression model, we estimated growth advantages for BA.4 and BA.5 of 0.08 (95% confidence interval (CI): 0.08-0.09) and 0.10 (95% CI: 0.09-0.11) per day, respectively, over BA.2 in South Africa. The continued discovery of genetically diverse Omicron lineages points to the hypothesis that a discrete reservoir, such as human chronic infections and/or animal hosts, is potentially contributing to further evolution and dispersal of the virus.