Preparation of (S)-epichlorohydrin using a novel halohydrin dehalogenase by selective conformation adjustment.

Chiral epichlorohydrin (ECH) is an attractive intermediate for chiral pharmaceuticals and chemicals preparation. The asymmetric synthesis of chiral ECH using 1,3-dicholoro-2-propanol (1,3-DCP) catalyzed by a haloalcohol dehalogenase (HHDH) was considered as a feasible approach. However, the reverse ring opening reaction caused low optical purity of chiral ECH, thus severely restricts the industrial application of HHDHs. In the present study, a novel selective conformation adjustment strategy was developed with an engineered HheCPS to regulate the kinetic parameters of the forward and reverse reactions, based on site saturation mutation and molecular simulation analysis. The HheCPS mutant E85P was constructed with a markable change in the conformation of (S)-ECH in the substrate pocket and a slight impact on the interaction between 1,3-DCP and the enzyme, which resulted in the kinetic deceleration of the reverse reactions. Compared with HheCPS, the catalytic efficiency (kcat(S)-ECH/Km(S)-ECH) of the reversed reaction dropped to 0.23-fold (from 0.13 to 0.03 mM-1 s-1), while the catalytic efficiency (kcat(1,3-DCP)/Km(1,3-DCP)) of the forward reaction only reduced from 0.83 to 0.71 mM-1 s-1. With 40 mM 1,3-DCP as substrate, HheCPS E85P catalyzed the synthesis of (S)-ECH with the yield up to 55.35% and the e.e. increased from 92.54 to >99%. Our work provided an effective approach for understanding the stereoselective catalytic mechanism as well as the green manufacturing of chiral epoxides.

Quality and flavor development of solid-state fermented surimi with Actinomucor elegans: A perspective on the impacts of carbon and nitrogen sources.

The influence of four carbon and nitrogen substrates on the quality and flavor of a novel surimi-based product fermented with Actinomucor elegans (A. elegans) was investigated, with a focus on carbon and nitrogen catabolite repression. The results showed that the substrate significantly affected mycelial growth, enzyme activities, and the metabolites of A. elegans. Although glucose significantly promoted A. elegans growth by 116.69%, it decreased enzyme secretion by 69.79% for α-amylase and 59.80% for protease, most likely by triggering the carbon catabolite repression pathway. Starch, soy protein, and wheat gluten substantially affected the textural properties of the fermented surimi. Furthermore, wheat gluten significantly promoted the protease activity (102.70%) and increased protein degradation during surimi fermentation. The fishy odor of surimi was alleviated through fermentation, and a correlation between the volatile compounds and A. elegans metabolism was observed. These results explore fermentation substrates in filamentous fungi metabolism from a catabolite repression perspective.

Effects of cluster nursing interventions on the prevention of pressure ulcers in intensive care units patients: A meta-analysis.

A meta-analysis was conducted to comprehensively evaluate the impact of cluster nursing interventions on the prevention of pressure ulcers (PUs) in intensive care unit (ICU) patients. Computer searches were performed in databases including Embase, Google Scholar, Cochrane Library, PubMed, Wanfang and China National Knowledge Infrastructure for randomized controlled trials (RCTs) implementing cluster nursing interventions for PUs prevention in ICU patients, with the search period covering the database inception to November 2023. Two researchers independently screened the literature, extracted data and conducted quality assessments. Stata 17.0 software was employed for data analysis. Overall, 17 RCTs involving 1463 ICU patients were included. The analysis showed that compared with conventional nursing, cluster nursing interventions significantly reduced the incidence of PUs (odds ratio: 0.24, 95% confidence intervals [CI]: 0.17-0.34, p < 0.001) and also significantly improved the levels of anxiety (standardized mean difference [SMD]: -1.39, 95% CI: -1.57 to 1.22, p < 0.001) and depression (SMD: -1.64, 95% CI: -2.02 to 1.26, p < 0.001) in ICU patients. This study indicates that the application of cluster nursing interventions in ICU patients can effectively reduce the incidence of PUs, as well as improve patients' anxiety and depression levels, thereby enhancing their quality of life, which is worth clinical promotion and application.

Plant Defensin-Dissimilar Thionin OsThi9 Alleviates Cadmium Toxicity in Rice Plants and Reduces Cadmium Accumulation in Rice Grains.

Thionins are important antibacterial peptides in plants. However, the roles of plant thionins, especially the defensin-dissimilar thionins, in alleviating heavy-metal toxicity and accumulation remain unclear. Here, cadmium (Cd)-related functions and mechanisms of the defensin-dissimilar rice thionin OsThi9 were investigated. OsThi9 was significantly upregulated in response to Cd exposure. OsThi9 was localized to the cell wall and was shown to bind Cd; these characters help to increase Cd tolerance. In Cd-exposed rice plants, OsThi9 overexpression significantly increased cell wall Cd binding, decreasing upward Cd translocation and subsequent Cd accumulation in shoots and straw, while OsThi9 knockout had inverse effects. Importantly, in rice plants grown in Cd-contaminated soils, OsThi9 overexpression significantly reduced Cd accumulation in brown rice (decrease of ≥ 51.8%) without negatively impairing the crop yield and essential elements. Thus, OsThi9 plays an important role in the alleviation of Cd toxicity and accumulation and has significant potential for developing low-Cd rice.

Validation of immunoassays for the Chlamydia trachomatis antigen Pgp3 using a chimeric monoclonal antibody.

Seroepidemiology, or measuring antibodies to pathogens to estimate population-level exposure, can provide useful public health data. The tests used, however, often lack sufficient validation data due to absence of a gold standard. For many pathogens, serum antibodies can be detected long after resolution of infection, but infection status is often used as a gold standard for antibody positivity. To ensure that recently developed antibody tests for seroepidemiology of Chlamydia trachomatis (Ct), the causative agent of urogenital chlamydia and the blinding eye disease trachoma, have high performance, we generated a chimeric antibody to the immunodominant Ct antigen Pgp3. Two clones were selected to evaluate the test performance of three assays to measure antibodies to Pgp3: multiplex bead assay (MBA), enzyme-linked immunosorbent assay (ELISA), and lateral flow assay (LFA). Overall, each assay demonstrated high accuracy and precision when tested using either clone, and the clones were stable when stored at - 20 °C and 4 °C for almost 2 years. The limit of detection was similar for MBA and LFA, but almost a log-fold higher (i.e. less sensitive) using ELISA. Overall, the chimeric antibodies represent stable control reagents for tests with robust performance and will facilitate deployment of these tests to other laboratories.

[Adaptive evolution of microorganisms based on industrial environmental perturbations].

The development of synthetic biology has greatly promoted the construction of microbial cell factories, providing an important strategy for green and efficient chemical production. However, the bottleneck of poor tolerance to harsh industrial environments has become the key factor hampering the productivity of microbial cells. Adaptive evolution is an important method to domesticate microorganisms for a certain period by applying targeted selection pressure to obtain desired phenotypic or physiological properties that are adapted to a specific environment. Recently, with the development of technologies such as microfluidics, biosensors, and omics analysis, adaptive evolution has laid the foundation for efficient productivity of microbial cell factories. Herein, we discuss the key technologies of adaptive evolution and their important applications in improvement of environmental tolerance and production efficiency of microbial cell factories. Moreover, we looked forward to the prospects of adaptive evolution to realize industrial production by microbial cell factories.

Investigation of Histamine Removal by Electrodialysis from the Fermented Fish Sauce and Its Effects on the Flavor.

Histamine is one of the most concerned safety indicators in fish sauce. Considering its charge property, electrodialysis (ED) was used to control the histamine in fish sauce, and studies were focused on three operating parameters: input current, pH, and flow velocity. A Box-Behnken design and response surface methodology was adopted to derive a statistical model, which indicated that 5.1 A input current, pH 3.8, and 40 L∙h-1 flow velocity were optimal operation conditions. Under this condition, the histamine removal rate reached 53.41% and the histamine content met the allowable histamine limit of below 400 mg·kg-1 in fish sauce, while the amino nitrogen (ANN) loss rate was only 15.46%. In addition, amino acids and volatile compounds changed differently during ED. As a result, with decreased histamine, the fish sauce after ED was also less salty and less fishy. The study first explored utilizing ED to remove histamine from fish sauce, which has positive implications for promoting the safety of aquatic products.

Transient receptor potential vanilloid subtype 1: A potential therapeutic target for fibrotic diseases.

The transient receptor potential vanilloid subtype 1 (TRPV1), belonging to the TRPV channel family, is a non-selective, calcium-dependent, cation channel implicated in several pathophysiological processes. Collagen, an extracellular matrix component, can accumulate under pathological conditions and may lead to the destruction of tissue structure, organ dysfunction, and organ failure. Increasing evidence indicates that TRPV1 plays a role in the development and occurrence of fibrotic diseases, including myocardial, renal, pancreatic, and corneal fibrosis. However, the mechanism by which TRPV1 regulates fibrosis remains unclear. This review highlights the comprehensive role played by TRPV1 in regulating pro-fibrotic processes, the potential of TRPV1 as a therapeutic target in fibrotic diseases, as well as the different signaling pathways associated with TRPV1 and fibrosis.

Regulating substrate preference of phosphatase by reshaping the "cap domain" for multi-enzymatic biosynthesis of high-purity monosaccharides.

Phosphatases are a class of enzymes catalyzing the cleavage of monophosphate ester bonds from the phosphorylated substrates. They have important applications in construction of in vitro multi-enzymatic system for monosaccharides. However, the enzymes generally show substrate ambiguity, which has become a bottleneck for efficient biosynthesis of target products with high purity. In this study, semirational design was performed on phosphatase from Thermosipho atlanticus (Ta-PST). The hotspot amino acid residues forming a "cap domain" were identified and selected for saturation mutagenesis. The mutant F179T and F179M showed improved substrate preference toward fructose-6-phosphate and mannose-6-phosphate, respectively. Coupling with other enzymes involved in the multi-enzymatic system under optimized conditions, the application of F179T led to fructose yield of 80% from 10 g/L maltodextrin and the ratio between the target product and by-product glucose was increased from 2:1 to 19:1. On the other hand, the application of F179M led to mannose yield of 59% with ratio of mannose to the by-products glucose and fructose increased from 1:1:1 to 14:2:1. Moreover, the molecular understanding of the beneficial substitution was gained by structural analysis and molecular dynamic simulations, giving important guidance to regulate the enzyme's substrate preference.

Improvement of multicatalytic properties of nitrilase from Paraburkholderia graminis for efficient biosynthesis of 2-chloronicotinic acid.

Nitrilases are promising biocatalysts to produce high-value-added carboxylic acids through hydrolysis of nitriles. However, since the enzymes always show low activity and sometimes with poor reaction specificity toward 2-chloronicotinonitrile (2-CN), very few robust nitrilases have been reported for efficient production of 2-chloronicotinic acid (2-CA) from 2-CN. Herein, a nitrilase from Paraburkholderia graminis (PgNIT) was engineered to improve its catalytic properties. We identified the beneficial residues via computational analysis and constructed the mutant library. The positive mutants were obtained and the activity of the "best" mutant F164G/I130L/N167Y/A55S/Q260C/T133I/R199Q toward 2-CN was increased from 0.14 × 10-3  to 4.22 U/mg. Its reaction specificity was improved with elimination of hydration activity. Molecular docking and molecular dynamics simulation revealed that the conformational flexibility, the nucleophilic attack distance, as well as the interaction forces between the enzyme and substrate were the main reason alternating the catalytic properties of PgNIT. With the best mutant as biocatalyst, 150 g/L 2-CN was completely converted, resulting in 2-CA accumulated to 169.7 g/L. When the substrate concentration was increased to 200 g/L, 203.1 g/L 2-CA was obtained with yield of 85.7%. The results laid the foundation for industrial production of 2-CA with the nitrilase-catalyzed route.

[Construction and application of microbial cell factories for unnatural amino acids].

Unnatural amino acids are widely used in medicine, pesticide, material, and other industries and the green and efficient synthesis has attracted a lot of attention. In recent years, with the rapid development of synthetic biology, microbial cell factories have become a promising means for biosynthesis of unnatural amino acids. This study reviewed the construction and application of microbial cell factories for unnatural amino acid, including the synthetic pathway reconstruction, design/modification of key enzymes and their coordinated regulation with precursors, blocking of competitive alternative pathways, and construction of cofactor circulation systems. Meanwhile, on the basis of the new principles for designing the microbial cell factories, new biosynthetic pathways adapted to cells and the production environment, as well as new biomanufacturing system established based on cell adaptive evolution and intelligent fermentation regulation, we looked forward to the further construction and application of microbial cell factories for industrial bio-production.

Simultaneous determination of fourteen ß2-agonist enantiomers in food animal muscles by liquid chromatography coupled with tandem mass spectrometry.

Illegal drug residues in animal derived foods are closely related to human's life and health. Studies on illegal drug residues and the metabolism, such as ß2-agonists in animals have attracted more and more attention. In most cases, ß2-agonists are suppliedand used astheracemate. The metabolic process and distribution of the two enantiomers in animal tissues are different. Therefore, it is very necessary to develop a simple and fast method for chiral resolution of these drugs in animal tissues. In this paper, a reliable resolution and determination method was presented using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for fourteen enantiomers of seven ß2-agonist racemates, clenbuterol (CLE), salbutamol (SAL), cimaterol (CIM), terbutaline (TER), clorprenaline (CLO), tulobuterol (TUL), penbuterol (PEN) in pork, beef, and lamb muscle samples. The samples were added the internal standard solution (IS) and extracted in the alkaline medium with acetonitrile. The further sample purification was accomplished through MCX solid phase extraction cartridge. Chromatographic chiral separation was carried out on a VancoShell chiral column (100 mm × 4.6 mm, 2.7 µm) with an isocratic mobile phase consisting of methanol and 10 mmol mL-1 ammonium formate aqueous solution (85:15, v/v). Under the optimized conditions, the resolution (R) of CIM was 2.0, CLE and PEN were 1.5, the others were all greater than 1.0. Enantiomeric determination was performed in the positive electrospray ionization mode using multiple reaction monitoring (MRM). The correlation coefficient (r) in the range of 0.2-25.0 µg L-1 was above 0.993. The average recoveries at the three spiking levels ranged from 95.3% to 117.7% with the relative standard deviation (RSD) lower than 15%. The limit of detection (LOD) and the limit of quantification (LOQ) of ß2-agonist enantiomers was 0.2 µg kg-1 and 0.5 µg kg-1 respectively. The method was successfully applied in the analysis and evaluation of ß2-agonist enantiomers in positive food animal muscle samples, CLE, SAL, TEB and CIM enantiomers were detected. The concentrations of the corresponding enantiomers were in the range of 1.06-17.3 µg kg-1, the lowest enantiomer fraction (EF) value was 0.42, and the highest value was 0.69. The work is expected to provide a method for chiral separation and enantiomeric determination of the further study of pharmacology, toxicity and residue elimination of ß2-agonist enantiomers.

Rational Regulation of Reaction Specificity of Nitrilase for Efficient Biosynthesis of 2-Chloronicotinic Acid through a Single Site Mutation.

Nitrilase-catalyzed hydrolysis of 2-chloronicotinonitrile (2-CN) is a promising approach for the efficient synthesis of 2-chloronicotinic acid (2-CA). The development of nitrilase with ideal catalytic properties is crucial for the biosynthetic route with industrial potential. Herein, a nitrilase from Rhodococcus zopfii (RzNIT), which showed much higher hydration activity than hydrolysis activity, was designed for efficient hydrolysis of 2-CN. Two residues (N165 and W167) significantly affecting the reaction specificity were precisely identified. By tuning these two residues, a single mutation of W167G with abolished hydration activity and 20-fold improved hydrolysis activity was obtained. Molecular dynamics simulation and molecular docking revealed that the mutation generated a larger binding pocket, causing the substrate 2-CN to bind more deeply in the pocket and form a delocalized π bond between the residues W190 and Y196, which reduced the negative influence of steric hindrance and electron effect caused by chlorine substituent. With mutant W167G as biocatalyst, 100 mM 2-CN was exclusively converted into 2-CA within 16 h. The study provides useful guidance in nitrilase engineering for simultaneous improvement of reaction specificity and catalytic activity, which are highly desirable in value-added carboxylic acids production from nitriles hydrolysis. IMPORTANCE 2-CA is an important building block for agrochemicals and pharmaceuticals with a rapid increase in demand in recent years. It is currently manufactured from 3-cyanopyridine by chemical methods. However, during the final step of 2-CN hydrolysis under high temperature and strong alkaline conditions, the byproduct 2-CM was generated except for the target product, leading to low yield and tedious separation steps. Nitrilase-mediated hydrolysis is regarded as a promising alternative for 2-CA production, which proceeded under mild conditions. Nevertheless, nitrilase capable of efficient hydrolysis of 2-CN has not been reported because the enzymes showed either extremely low activity or surprisingly high hydration activity toward 2-CN. Herein, the reaction specificity of RzNIT was precisely tuned through a single site mutation. The mutant exhibited remarkably enhanced hydrolysis activity without the formation of byproducts, providing a robust biocatalyst for 2-CA biosynthesis with industrial potential.

High-throughput assay of tyrosine phenol-lyase activity using a cascade of enzymatic reactions.

Tyrosine phenol-lyase (TPL) exhibits great potential in industrial biosynthesis of l-tyrosine and its derivates. To uncover and screen TPLs with excellent catalytic properties, there is unmet demand for development of facile and reliable screening system for TPL. Here we presented a novel assay format for the detection of TPL activity based on catechol 2,3-dioxygenase (C23O)-catalyzed reaction. Catechol released from TPL-catalyzed cleavage of 3,4-dihydroxy-l-phenylalanine (l-DOPA) was further oxidized by C23O to form 2-hydroxymuconate semialdehyde, which could be readily detected by spectrophotometric measurements at 375 nm. The assay achieved a unique balance between the ease of operation and superiority of analytical performances including linearity, sensitivity and accuracy. In addition, this assay enabled real-time monitoring of TPL activity with high efficiency and reliability. As C23O is highly specific towards catechol, a non-natural product of microorganism, the assay was therefore accessible to both crude cell extracts and the whole-cell system without elaborate purification steps of enzymes, which could greatly expedite discovery and engineering of TPLs. This study provided fundamental principle for high-throughput screening of other enzymes consuming or producing catechol derivatives.

Development and Evaluation of a TaqMan Real-Time PCR Assay for the Rapid Detection of Cross-Contamination of RD (Human) and L20B (Mouse) Cell Lines Used in Poliovirus Surveillance.

BACKGROUND: The cross-contamination of cell lines in culture is a persistent problem. Genetically modified L20B (Mouse) and RD (Human Rhabdomyosarcoma) cell lines are commonly used in poliovirus research, surveillance, and diagnostics. Cross-contamination between these cell lines leads to unreproducible results and unreliable surveillance data, negatively affecting public health. The gold standard method for cell authentication is Short Tandem Repeats analysis, which is time-consuming and expensive. The disadvantage of STR is limited detection of interspecies contamination. METHODS: This assay targets the mitochondrial cytochrome c oxidase subunit I (MTCO1) gene, a highly conserved and emergent DNA barcode region for detection of cross-contamination in RD and L20B cell lines. The MagNA Pure Compact instrument and ABI 7500 Fast Dx Real-time PCR systems were used for DNA extraction and to perform real-time PCR respectively. RESULTS: The newly developed assay is very sensitive with a limit of detection of 100 RD cells/1 million L20B/mL. The amplification efficiency and R2-value were 102.26% and 0.9969 respectively. We evaluated specificity of the assay with five human and four mouse cell lines, as well as monkey and rat cell lines. The assay showed no cross-reactivity with genomic DNA from human, mouse, rat, or monkey cell lines. The analytical sensitivity was also evaluated by spiking varying amounts of RD cells (0.001% - 10%) into L20B cells. There was no difference in CT values when running single-plex or duplex PCR reactions with similar experimental conditions. CONCLUSIONS: We have developed and validated a TaqMan real-time PCR assay, a sensitive method for the detection of cross-contamination of RD and L20B cell lines.

Atypical endometrial hyperplasia in a 35-year-old woman: A case report and literature review.

BACKGROUND: Atypical endometrial hyperplasia (AEH) is a common precancerous lesion of endometrial carcinoma (EC). The risk factors for AEH and EC directly or indirectly related to estrogen exposure include early menarche, nulliparity, polycystic ovarian syndrome, diabetes, and obesity. Both AEH and EC rarely occur in young patients (< 40-years-old), who may desire to maintain their fertility. Evaluating the cancer risk of AEH patients is helpful for the determination of therapeutic plans. CASE SUMMARY: We report a rare case of AEH in a 35-year-old woman who presented to the Hunan Provincial Maternal and Child Health Care Hospital with a large mass in the uterus. She married at 20-years-old, and had been married for more than 15 years to date. Several characteristics of this patient were observed, including nulliparity, limited sexual activity (intercourse 1-2 times a year) in recent years, and irregular vaginal bleeding for 2 years. Gynecological examination revealed an enlarged uterus, similar to the uterus size in the fourth month of pregnancy, and the uterine wall was relatively hard. Curettage was performed based on transvaginal sonography and magnetic resonance imaging results. Findings from the pathological examination were typical for AEH. The patient was cured after treatment with the standard therapy of high-dose progesterone. CONCLUSION: In patients with intrauterine lumps that may be malignant, a pathological report should be obtained.

Analysis of the initial lot of the CDC 2019-Novel Coronavirus (2019-nCoV) real-time RT-PCR diagnostic panel.

At the start of the COVID-19 pandemic, the Centers for Disease Control and Prevention (CDC) designed, manufactured, and distributed the CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel for SARS-CoV-2 detection. The diagnostic panel targeted three viral nucleocapsid gene loci (N1, N2, and N3 primers and probes) to maximize sensitivity and to provide redundancy for virus detection if mutations occurred. After the first distribution of the diagnostic panel, state public health laboratories reported fluorescent signal in the absence of viral template (false-positive reactivity) for the N3 component and to a lesser extent for N1. This report describes the findings of an internal investigation conducted by the CDC to identify the cause(s) of the N1 and N3 false-positive reactivity. For N1, results demonstrate that contamination with a synthetic template, that occurred while the "bulk" manufactured materials were located in a research lab for quality assessment, was the cause of false reactivity in the first lot. Base pairing between the 3' end of the N3 probe and the 3' end of the N3 reverse primer led to amplification of duplex and larger molecules resulting in false reactivity in the N3 assay component. We conclude that flaws in both assay design and handling of the "bulk" material, caused the problems with the first lot of the 2019-nCoV Real-Time RT-PCR Diagnostic Panel. In addition, within this study, we found that the age of the examined diagnostic panel reagents increases the frequency of false positive results for N3. We discuss these findings in the context of improvements to quality control, quality assurance, and assay validation practices that have since been improved at the CDC.

Recent advancements in enzyme engineering via site-specific incorporation of unnatural amino acids.

With increased attention to excellent biocatalysts, evolving methods based on nature or unnatural amino acid (UAAs) mutagenesis have become an important part of enzyme engineering. The emergence of powerful method through expanding the genetic code allows to incorporate UAAs with unique chemical functionalities into proteins, endowing proteins with more structural and functional features. To date, over 200 diverse UAAs have been incorporated site-specifically into proteins via this methodology and many of them have been widely exploited in the field of enzyme engineering, making this genetic code expansion approach possible to be a promising tool for modulating the properties of enzymes. In this context, we focus on how this robust method to specifically incorporate UAAs into proteins and summarize their applications in enzyme engineering for tuning and expanding the functional properties of enzymes. Meanwhile, we aim to discuss how the benefits can be achieved by using the genetically encoded UAAs. We hope that this method will become an integral part of the field of enzyme engineering in the future.

High-throughput quantitation of SARS-CoV-2 antibodies in a single-dilution homogeneous assay.

SARS-CoV-2 emerged in late 2019 and has since spread around the world, causing a pandemic of the respiratory disease COVID-19. Detecting antibodies against the virus is an essential tool for tracking infections and developing vaccines. Such tests, primarily utilizing the enzyme-linked immunosorbent assay (ELISA) principle, can be either qualitative (reporting positive/negative results) or quantitative (reporting a value representing the quantity of specific antibodies). Quantitation is vital for determining stability or decline of antibody titers in convalescence, efficacy of different vaccination regimens, and detection of asymptomatic infections. Quantitation typically requires two-step ELISA testing, in which samples are first screened in a qualitative assay and positive samples are subsequently analyzed as a dilution series. To overcome the throughput limitations of this approach, we developed a simpler and faster system that is highly automatable and achieves quantitation in a single-dilution screening format with sensitivity and specificity comparable to those of ELISA.

Rapid development of neutralizing and diagnostic SARS-COV-2 mouse monoclonal antibodies.

The need for high-affinity, SARS-CoV-2-specific monoclonal antibodies (mAbs) is critical in the face of the global COVID-19 pandemic, as such reagents can have important diagnostic, research, and therapeutic applications. Of greatest interest is the ~ 300 amino acid receptor binding domain (RBD) within the S1 subunit of the spike protein because of its key interaction with the human angiotensin converting enzyme 2 (hACE2) receptor present on many cell types, especially lung epithelial cells. We report here the development and functional characterization of 29 nM-affinity mouse SARS-CoV-2 mAbs created by an accelerated immunization and hybridoma screening process. Differing functions, including binding of diverse protein epitopes, viral neutralization, impact on RBD-hACE2 binding, and immunohistochemical staining of infected lung tissue, were correlated with variable gene usage and sequence.