Omicron COVID-19 Case Estimates Based on Previous SARS-CoV-2 Wastewater Load, Regional Municipality of Peel, Ontario, Canada.

We determined correlations between SARS-CoV-2 load in untreated water and COVID-19 cases and patient hospitalizations before the Omicron variant (September 2020-November 2021) at 2 wastewater treatment plants in the Regional Municipality of Peel, Ontario, Canada. Using pre-Omicron correlations, we estimated incident COVID-19 cases during Omicron outbreaks (November 2021-June 2022). The strongest correlation between wastewater SARS-CoV-2 load and COVID-19 cases occurred 1 day after sampling (r = 0.911). The strongest correlation between wastewater load and COVID-19 patient hospitalizations occurred 4 days after sampling (r = 0.819). At the peak of the Omicron BA.2 outbreak in April 2022, reported COVID-19 cases were underestimated 19-fold because of changes in clinical testing. Wastewater data provided information for local decision-making and are a useful component of COVID-19 surveillance systems.

A mechanism-based inactivator of glycoside hydrolases involving formation of a transient non-classical carbocation.

The design of mechanism-based enzyme inactivators to generate chemical probes for biological research is an important challenge in carbohydrate chemistry. Here we describe the synthesis and biological evaluation of a novel carbocyclic mechanism-based inactivator of galactosidases (glycoside hydrolase families 27 and 36). Upon catalysis of this unnatural substrate, a transient non-classical carbocation forms within the enzyme active site. We show that the inactivation event, which proceeds via a bicyclobutonium ion intermediate, leads to a single alkylation event that occurs on the enzymatic nucleophile, an aspartic acid residue in this case. We also show that the catalytic proficiencies for enzymatic hydrolysis of substrates and inactivation by our bicyclo[4.1.0]heptyl analogue of galactose differ by only a factor of 20. This inactivator has the potential for further development as a useful biological research tool for both basic research and biotechnological applications.

Neuraminidase substrate promiscuity permits a mutant Micromonospora viridifaciens enzyme to synthesize artificial carbohydrates.

Mutation of the nucleophilic amino acid residue tyrosine to the small nonpolar residue glycine (Y370G) in the active site of Micromonospora viridifaciens neuraminidase (MvNA) produces an efficient catalyst for the transfer of N-acetylneuraminic acid from an artificial substrate (i.e., phenyl N-acetyl-ß-D-neuraminide) to a sugar acceptor (e.g., D-lactose, D-glucose, D-mannose, D-raffinose, D-allose, or D-fructose) to give N-acetyl-α-neuraminide coupled carbohydrate products. In addition, this mutant enzyme (MvNA Y370G) catalyzes the transfer of a sugar residue from the artificial substrate 2-fluorophenyl N-acetyl-ß-D-neuraminide to methyl glycopyranoside acceptors. Interestingly, when trans-glycosylation reactions are conducted in aqueous solutions containing 30% (v/v) acetonitrile, the α-anomeric acceptors of methyl glucopyranoside and galactopyranoside generate higher product yields than do their corresponding ß-anomers. Specifically, a 64 h reaction with 2-fluorophenyl N-acetyl-ß-D-neuraminide as the limiting reagent and the acceptors methyl α-d-galactopyranoside, methyl α-D-glucopyranoside, or methyl α-D-mannopyranoside gives trans-glycosylation product yields of 22%, 31%, or 34%, respectively. With methyl α-D-galactopyranoside as the acceptor, trans-glycosylations catalyzed by both MvNA Y370G and a 2,6-sialyltransferase yield identical products, which we identified as methyl N-acetyl-α-D-neuraminyl-(2 → 6)-α-D-galactopyranoside. The MvNA Y370G-catalyzed coupling of N-acetylneuraminic acid to these three methyl α-d-glycopyranoside acceptors is favored by factors of 18­27-fold over the competing hydrolysis reaction. These coupling efficiencies likely arise from nonselective interactions between the acceptor glycopyranoside and MvNA Y370G, which preferentially places a carbohydrate hydroxyl group rather than water in close proximity to the active site where this functionality intercepts the nascent neuraminyl oxacarbenium ion that is formed during cleavage of the glycosidic bond in the aryl N-acetyl-ß-D-neuraminide donor. The ability to transfer N-acetylneuraminic acid from a stable and readily accessible donor to acceptor carbohydrates that are not substrates for sialyltransferases is one step on the path for the production of pseudohuman glycoproteins from nonmammalian cell lines.

First-time fathers' experiences and needs during pregnancy and childbirth: a descriptive qualitative study.

OBJECTIVES: to explore first-time fathers' experiences and needs during their wives' pregnancy and childbirth in Singapore. DESIGN AND SETTING: a descriptive qualitative was conducted. Participants were recruited from two obstetric wards in a tertiary hospital in Singapore from November to December 2012. Semi-structured, face-to-face interviews were used to collect data and themes from the interviews were generated using thematic analysis. PARTICIPANTS: a purposive sample of 16 first-time fathers aged above 21 years who accompanied their wives throughout pregnancy and childbirth were recruited from the postnatal wards between one to three days after the birth of their children. FINDINGS: four themes emerged from 16 subthemes: (1) Emotional changes experienced; (2) Adaptive and supportive behaviours adopted; (3) Social support received and (4) Suggestions for improvement to the current maternity care. First-time fathers experienced a range of emotions from being happy and excited to feeling shocked and worried and to feeling calm. Adaptive and supportive behaviours were adopted to deal with the pregnancy changes and better support their wives. In the course of their transition to fatherhood, they also received support from their family, friends, workplaces and the health care professionals. Fathers suggested more information, timely, empathetic and professional care be given and a review to the current administrative/logistical policies. CONCLUSIONS: all fathers modified their behaviours for the sake of protecting their wives and unborn children. Support from their family, friends, workplaces and the health care professionals was invaluable and greatly appreciated. IMPLICATIONS FOR PRACTICE: health care professionals can guide and support fathers by providing them with more information and preparing them for the unknown changes. Future studies are needed to develop intervention programme for fathers to improve their experiences and adaptive behaviours.

Mechanistic evaluation of MelA α-galactosidase from Citrobacter freundii: a family 4 glycosyl hydrolase in which oxidation is rate-limiting.

The MelA gene from Citrobacter freundii, which encodes a glycosyl hydrolase family 4 (GH4) α-galactosidase, has been cloned and expressed in Escherichia coli. The recombinant enzyme catalyzes the hydrolysis of phenyl α-galactosides via a redox elimination-addition mechanism involving oxidation of the hydroxyl group at C-3 and elimination of phenol across the C-1-C-2 bond to give an enzyme-bound glycal intermediate. For optimal activity, the MelA enzyme requires two cofactors, NAD(+) and Mn(2+), and the addition of a reducing agent, such as mercaptoethanol. To delineate the mechanism of action for this GH4 enzyme, we measured leaving group effects, and the derived ß(lg) values on V and V/K are indistinguishable from zero (-0.01 ± 0.02 and 0.02 ± 0.04, respectively). Deuterium kinetic isotope effects (KIEs) were measured for the weakly activated substrate phenyl α-D-galactopyranoside in which isotopic substitution was incorporated at C-1, C-2, or C-3. KIEs of 1.06 ± 0.07, 0.91 ± 0.04, and 1.02 ± 0.06 were measured on V for the 1-(2)H, 2-(2)H, and 3-(2)H isotopic substrates, respectively. The corresponding values on V/K were 1.13 ± 0.07, 1.74 ± 0.06, and 1.74 ± 0.05, respectively. To determine if the KIEs report on a single step or on a virtual transition state, we measured KIEs using doubly deuterated substrates. The measured (D)V/K KIEs for MelA-catalyzed hydrolysis of phenyl α-D-galactopyranoside on the dideuterated substrates, (D)V/K((3-D)/(2-D,3-D)) and (D)V/K((2-D)/(2-D,3-D)), are 1.71 ± 0.12 and 1.71 ± 0.13, respectively. In addition, the corresponding values on V, (D)V((3-D)/(2-D,3-D)) and (D)V((2-D)/(2-D,3-D)), are 0.91 ± 0.06 and 1.01 ± 0.06, respectively. These observations are consistent with oxidation at C-3, which occurs via the transfer of a hydride to the on-board NAD(+), being concerted with proton removal at C-2 and the fact that this step is the first irreversible step for the MelA α-galactosidase-catalyzed reactions of aryl substrates. In addition, the rate-limiting step for V(max) must come after this irreversible step in the reaction mechanism.

Functional and sequence analysis of human neuroglobin gene promoter region.

Neuroglobin (Ngb), a recently found oxygen-binding protein belonging to the vertebrate globin family, is mainly expressed in neurons of brains and eyes. Current studies have revealed diverse potential functions of Ngb and it was found to be able to reduce the severity of stroke and Alzheimer's disease, implying its importance in brains. However, the mechanism of Ngb regulation of transcription has not been elucidated yet. In this study, we analyzed the 5'-flanking region of human neuroglobin gene (NGB) and identified a transcription start site (TSS) located at -306bp relative to the translation start site ATG. We characterized the proximal promoter of NGB and found two GC-boxes located at -16 and +30bp relative to the TSS which are bound by transcription factor Sp1 and Sp3. Mutation of either GC-box led to a significant reduction in NGB promoter activity, while overexpression of Sp1 and Sp3 resulted in activation of the promoter. However, two putative NRSE sites (-359 and -127bp relative to the TSS) apparently showed no influence on NGB tissue-specific expression. Treatment of two non-neuronal cell lines HeLa and BEAS-2B with 5-aza-2'-deoxycytidine remarkably induced NGB expression, suggesting a potential role of DNA methylation in regulating NGB tissue-specific expression.

Brønsted analysis of an enzyme-catalyzed pseudo-deglycosylation reaction: mechanism of desialylation in sialidases.

The Micromonospora viridifaciens Y370G inverting mutant sialidase has been found to possess beta-sialidase activity with various fluoro-substituted phenyl beta-sialosides. A reagent panel of seven mono- and difluorophenyl beta-d-sialosides was synthesized, and these compounds were used, in conjunction with the parent phenyl beta-d-sialoside, to probe the mechanism of M. viridifaciens Y370G mutant sialidase-catalyzed hydrolyses. These hydrolysis reactions mimic the deglycosylation reaction step of the crucial tyrosinyl enzyme-bound intermediate that is formed during the corresponding wild-type sialidase reactions. The derived Brønsted parameter (beta(lg)) on k(cat)/K(m) is -0.46 +/- 0.02 for the four substrates that display significant activity, and these span a range of leaving group abilities (as judged by the pK(a) of their conjugate acids being between 7.09 and 9.87). The 4-fluoro, 2,3- and 2,5-difluorosubstrates display a diminished activity, whereas the 3,5-difluoro compound undergoes catalyzed hydrolysis exceedingly slowly. These observations, taken with solvent deuterium kinetic isotope effects (k(H(2))(O)/k(D(2))(O)) on the catalyzed hydrolysis of the 2-fluorophenyl substrate of 0.88 +/- 0.24 (k(cat)/K(m)) and 1.16 +/- 0.12 (k(cat)) and the poor inhibition shown by phenol (IC(50) > 1 mM), are consistent with glycosidic C-O cleavage being rate determining for both k(cat)/K(m) and k(cat) with little or no protonation of the departing aryloxide leaving group. The kinetic data reported herein are consistent with rate-limiting glycoside hydrolysis occurring via two distinct transition states that incorporates a nonproductive binding component for the tighter binding substrates.

[Effect of ventral prostate secretory proteins on oviductal fluid glycoproteins in golden hamsters].

OBJECTIVE: To investigate the effect of the secretory proteins of the ventral prostate on the glycoproteins in the oviductal fluid of golden hamsters. METHODS: Male golden hamsters were divided into four groups: sham operation (SH), total removal of accessory sex glands (TX), and retainment of the ventral prostate only (VP). Oviductal fluid was collected from female hamsters at 0.5, 2, 4 and 6 h after mating with the males of different operated groups with or without ventral prostate. Glycoproteins were probed with a panel of lectins and their changes in the oviductal fluid were analyzed by Western blot. RESULTS: The 47 000, 52 000, 81 000 and 128 000 WGA-binding proteins were observed in the oviductal fluid of the 6 h TX group, the 32 000, 35 500, 47 000 and 52 000 WGA-binding glycoproteins noted in the 6 h VP group, the 47 000, 68 000, 95 000 and 128 000 pisum sativum agglutinin (PSA)-binding glycoproteins shown in the 6 h TX and VP groups, two extra 32 000 and 37 500 bands detected in the 6 h VP group, the 47 000 and 52 000 dolichos biflorus agglutinin (DBA)-binding glycoproteins present in the 6 h VP but absent in the 6 h TX group. CONCLUSION: Ventral prostate secretory proteins affect acetylglucosamine, N-acetylgalactosamine/galactose and mannose in the oviductal fluid collected 6 hours after mating. And these glycoproteins may play an important role in the development of embryos.

[Identification of sperm-binding proteins in the ventral prostate of the golden hamster].

OBJECTIVE: To investigate the binding of secretory proteins in the ventral prostate to the surface of sperm. METHODS: We used different techniques to demonstrate the possibility of ventral prostate secretory proteins binding to sperm in golden hamsters. Polyclonal antibodies against crude secretion of the ventral prostate cultured in rabbits were used to detect the antigens in hamster epididymal, uterine and oviductal spermatozoa by indirect immunofluorescence technique. The uterine and oviductal spermatozoa were collected after mating with the males with or without ventral prostate glands. The ventral prostate secretory proteins were isolated and transblotted to the membrane, which was incubated with the biotinylated epididymal sperm membrane proteins, and then the biotinylated binding proteins were stained. RESULTS: An immunoreaction restricted to the middle piece was observed in the sperm incubated with the ventral prostate secretion and ejaculated sperm recovered from the uteri and oviducts. The rate of the epididymal sperm bound with the ventral prostate secretory proteins was (80 +/- 5) %, and the rats of the sperm binding to the ventral prostate secretory proteins were (30.0 +/- 4.6) % from the uterus and (16.0 +/- 3.6) % from the oviduct after mating with the males with ventral prostate glands, significantly higher than after mating with those without prostate glands (P < 0.01). Five bands were identified by Western blot analysis in vitro of the ventral prostate secretory proteins incubated with biotinylated epididymal sperm membrane proteins. CONCLUSION: The present data indicate that ventral prostate secretory proteins bind to the middle piece of sperm in golden hamsters.

The hydrolase and transferase activity of an inverting mutant sialidase using non-natural beta-sialoside substrates.

The Y370G inverting mutant sialidase from Micromonospora viridifaciens possesses beta-sialidase activity with phenyl beta-sialoside (Ph-betaNeuAc) to give alpha-sialic acid as the first formed product. The derived catalytic rate constants for k(cat) and k(cat)/K(m) are 13.3 +/- 0.3 and (2.9 +/- 0.3) x 10(5) M(-)(1) s(-)(1), respectively. This enzyme is highly specific for the phenyl substrate, with substituted phenyl and thiophenyl leaving groups having k(cat) values that are at least 1000-fold lower. In addition, the Y370G mutant can transfer the sialic acid moiety from Ph-betaNeuAc to lactose in yields of up to 13%. Greater than 90% of the sialyl-lactose product formed in the coupling reactions is the alpha-2,6-isomer. A library encoding 6 x 10(5) different sialidases was constructed by mutating Y370, E260, T309, N310, and N311, residues that include and are proximal the catalytic tyrosine residue. A total of 2628 individuals were screened for hydrolytic activity against 4-nitrophenyl 2-thio-beta-sialoside and 4-methylumbelliferyl beta-sialoside. However, none of the mutants screened possessed a significant activity against either of the beta-sialosides.

Detecting exon deletions and duplications of the DMD gene using Multiplex Ligation-dependent Probe Amplification (MLPA).

OBJECTIVES: To evaluate the efficacy of Multiplex Ligation-dependent Probe Amplification (MLPA) technique in comparison with the traditional multiplex PCR assay in detection of exon deletions and duplications of the DMD gene. DESIGN AND METHODS: The sensitivity and accuracy of MLPA were assessed and compared with the multiplex PCR in a total of 63 subjects including 43 subjects with Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD) and 20 female carriers. RESULTS: MLPA was able to detect all the known deletions and duplications; it detected four additional mutations that had been missed by multiplex PCR. In addition, the extent of the deletions and duplications could be more accurately defined which in turn facilitated a genotype-phenotype correlation. CONCLUSIONS: MLPA is superior to multiplex PCR. It should be the method of choice for the detection of exon deletions and duplications of the DMD gene in patients with DMD or BMD, as well as in female carriers.

Male genital tract antioxidant enzymes: their source, function in the female, and ability to preserve sperm DNA integrity in the golden hamster.

Recently, we reported that male accessory sex gland (ASG) secretions protect sperm genomic integrity by demonstrating that DNA damage was more extensive in sperm not exposed to the secretions. The present study was conducted to find out if ASGs secrete the main antioxidant enzymes superoxide dismutase (SOD), glutathione peroxidase (GPx or GSH-Px), and catalase (CAT) and if the most abundant one, SOD, can protect those sperm that were not exposed to ASG secretions against NADPH-induced oxidative stress. Four experimental groups of male golden hamsters were used: intact animals with proven fertility, animals with all major ASGs removed (TX), animals that were bilaterally vasectomized, and sham-operated controls. SOD, CAT, and GPx activities were measured in secretions from all 5 ASGs and sperm-free uterine flushing from virgin females and those mated with the experimental males. The alkaline comet assay was used to analyze DNA integrity of the TX group sperm after incubation in a medium containing 50 U/mL of SOD along with 0 to 20 mmol/L NADPH. The main antioxidant enzyme in ASGs was SOD from coagulating glands (P <.05) and GPx together with CAT from ampullary glands (P <.05). Uterine flushing of ejaculates that contained ASG secretions had more SOD and CAT activities than those with epididymal secretions alone (P <.05 and P <.001, respectively), whereas activity of GPx was the same (P >.05). Addition of SOD in vitro dose dependently decreased the incidence of single-strand DNA damage in sperm not exposed to ASG secretions incubated in the presence of 0 to 20 mmol/L NADPH (P <.001). These results indicated that, in terms of abundance, SOD was the main antioxidant enzyme secreted by male ASGs, whereas CAT was the second one. The GPx activity came from both epididymis and ASGs. We conclude that ASG secretions play a significant role in protecting sperm against oxidative stress.