Divergent Biochemical Properties and Disparate Impact of Arrhythmogenic Calmodulin Mutations on Zebrafish Cardiac Function.

Calmodulin (CaM) is a ubiquitous, small cytosolic calcium (Ca2+)-binding sensor that plays a vital role in many cellular processes by binding and regulating the activity of over 300 protein targets. In cardiac muscle, CaM modulates directly or indirectly the activity of several proteins that play a key role in excitation-contraction coupling (ECC), such as ryanodine receptor type 2 (RyR2),  l-type Ca2+ (Cav1.2), sodium (NaV1.5) and potassium (KV7.1) channels. Many recent clinical and genetic studies have reported a series of CaM mutations in patients with life-threatening arrhythmogenic syndromes, such as long QT syndrome (LQTS) and catecholaminergic polymorphic ventricular tachycardia (CPVT). We recently showed that four arrhythmogenic CaM mutations (N98I, D132E, D134H, and Q136P) significantly reduce the binding of CaM to RyR2. Herein, we investigate in vivo functional effects of these CaM mutations on the normal zebrafish embryonic heart function by microinjecting complementary RNA corresponding to CaMN98I, CaMD132E, CaMD134H, and CaMQ136P mutants. Expression of CaMD132E and CaMD134H mutants results in significant reduction of the zebrafish heart rate, mimicking a severe form of human bradycardia, whereas expression of CaMQ136P results in an increased heart rate mimicking human ventricular tachycardia. Moreover, analysis of cardiac ventricular rhythm revealed that the CaMD132E and CaMN98I zebrafish groups display an irregular pattern of heart beating and increased amplitude in comparison to the control groups. Furthermore, circular dichroism spectroscopy experiments using recombinant CaM proteins reveals a decreased structural stability of the four mutants compared to the wild-type CaM protein in the presence of Ca2+. Finally, Ca2+-binding studies indicates that all CaM mutations display reduced CaM Ca2+-binding affinities, with CaMD132E exhibiting the most prominent change. Our data suggest that CaM mutations can trigger different arrhythmogenic phenotypes through multiple and complex molecular mechanisms.

Demonstrating the reliability of in vivo metabolomics based chemical grouping: towards best practice.

While grouping/read-across is widely used to fill data gaps, chemical registration dossiers are often rejected due to weak category justifications based on structural similarity only. Metabolomics provides a route to robust chemical categories via evidence of shared molecular effects across source and target substances. To gain international acceptance, this approach must demonstrate high reliability, and best-practice guidance is required. The MetAbolomics ring Trial for CHemical groupING (MATCHING), comprising six industrial, government and academic ring-trial partners, evaluated inter-laboratory reproducibility and worked towards best-practice. An independent team selected eight substances (WY-14643, 4-chloro-3-nitroaniline, 17α-methyl-testosterone, trenbolone, aniline, dichlorprop-p, 2-chloroaniline, fenofibrate); ring-trial partners were blinded to their identities and modes-of-action. Plasma samples were derived from 28-day rat tests (two doses per substance), aliquoted, and distributed to partners. Each partner applied their preferred liquid chromatography-mass spectrometry (LC-MS) metabolomics workflows to acquire, process, quality assess, statistically analyze and report their grouping results to the European Chemicals Agency, to ensure the blinding conditions of the ring trial. Five of six partners, whose metabolomics datasets passed quality control, correctly identified the grouping of eight test substances into three categories, for both male and female rats. Strikingly, this was achieved even though a range of metabolomics approaches were used. Through assessing intrastudy quality-control samples, the sixth partner observed high technical variation and was unable to group the substances. By comparing workflows, we conclude that some heterogeneity in metabolomics methods is not detrimental to consistent grouping, and that assessing data quality prior to grouping is essential. We recommend development of international guidance for quality-control acceptance criteria. This study demonstrates the reliability of metabolomics for chemical grouping and works towards best-practice.

Egg Activation at Fertilization by a Soluble Sperm Protein.

The most fundamental unresolved issue of fertilization is to define how the sperm activates the egg to begin embryo development. Egg activation at fertilization in all species thus far examined is caused by some form of transient increase in the cytoplasmic free Ca(2+) concentration. What has not been clear, however, is precisely how the sperm triggers the large changes in Ca(2+) observed within the egg cytoplasm. Here, we review the studies indicating that the fertilizing sperm stimulates a cytosolic Ca(2+) increase in the egg specifically by delivering a soluble factor that diffuses into the cytosolic space of the egg upon gamete membrane fusion. Evidence is primarily considered in species of eggs where the sperm has been shown to elicit a cytosolic Ca(2+) increase by initiating Ca(2+) release from intracellular Ca(2+) stores. We suggest that our best understanding of these signaling events is in mammals, where the sperm triggers a prolonged series of intracellular Ca(2+) oscillations. The strongest empirical studies to date suggest that mammalian sperm-triggered Ca(2+) oscillations are caused by the introduction of a sperm-specific protein, called phospholipase C-zeta (PLCζ) that generates inositol trisphosphate within the egg. We will discuss the role and mechanism of action of PLCζ in detail at a molecular and cellular level. We will also consider some of the evidence that a soluble sperm protein might be involved in egg activation in nonmammalian species.

Quantitative Analysis of The High-Yield Hydrolysis of Kelp by Laminarinase and Alginate Lyase.

Kelp is an abundant, farmable biomass-containing laminarin and alginate as major polysaccharides, providing an excellent model substrate to study their deconstruction by simple enzyme mixtures. Our previous study showed strong reactivity of the glycoside hydrolase family 55 during hydrolysis of purified laminarin, raising the question of its reactivity with intact kelp. In this study, we determined that a combination of a single glycoside hydrolase family 55 ß-1,3-exoglucanase with a broad-specificity alginate lyase from the polysaccharide lyase family 18 gives efficient hydrolysis of untreated kelp to a mixture of simple sugars, that is, glucose, gentiobiose, mannitol-end glucose, and mannuronic and guluronic acids and their soluble oligomers. Quantitative assignments from nanostructure initiator mass spectrometry (NIMS) and 2D HSQC NMR spectroscopy and analysis of the reaction time-course are provided. The data suggest that binary combinations of enzymes targeted to the unique polysaccharide composition of marine biomass are sufficient to deconstruct kelp into soluble sugars for microbial fermentation.

Impaired Binding to Junctophilin-2 and Nanostructural Alteration in CPVT Mutation.

[Figure: see text].

Time for change? Feasibility of introducing micromodules into medical student education: a randomised controlled trial.

INTRODUCTION: Didactic lectures have been the foundation of learning for many medical students. However, in recent years, the flipped classroom model has become increasingly popular in medical education. This approach enhances pre-class learning, allowing the limited contact time between clinicians and medical students to be focused on practical issues. This study evaluated the effectiveness and non-inferiority of online micromodule teaching in terms of knowledge transfer concerning specific urology topics. METHODS: Medical students without prior exposure to the urology subspecialty were enrolled in the study, then randomised to a traditional didactic lecture group or an online micromodule group. Knowledge transfer was assessed by pre-intervention and post-intervention multiple-choice questions and objective structured clinical examinations that involved the acquisition of medical histories from real patients. RESULTS: In total, 45 medical students were enrolled (22 in the traditional didactic group and 23 in the online micromodule group). In terms of knowledge transfer (assessed by objective structured clinical examinations), the efficacy of online micromodules was comparable to traditional didactic lectures, although the difference was not statistically significant (P=0.823). There were no significant differences in terms of knowledge acquisition, retention, or clinical application between the two groups. CONCLUSION: In terms of acquiring, retaining, and applying foundational urological knowledge, online micromodules can help medical students to achieve outcomes comparable with the outcomes of didactic lectures. Online micromodules may be a viable alternative to traditional didactic lectures in urology education.

Arrhythmia-Associated Calmodulin E105A Mutation Alters the Binding Affinity of CaM to a Ryanodine Receptor 2 CaM-Binding Pocket.

Calmodulin (CaM) is a small, multifunctional calcium (Ca2+)-binding sensor that binds and regulates the open probability of cardiac ryanodine receptor 2 (RyR2) at both low and high cytosolic Ca2+ concentrations. Recent isothermal titration calorimetry (ITC) studies of a number of peptides that correspond to different regions of human RyR2 showed that two regions of human RyR2 (3584-3602aa and 4255-4271aa) bind with high affinity to CaM, suggesting that these two regions might contribute to a putative RyR2 intra-subunit CaM-binding pocket. Moreover, a previously characterized de novo long QT syndrome (LQTS)-associated missense CaM mutation (E105A) which was identified in a 6-year-old boy, who experienced an aborted first episode of cardiac arrest revealed that this mutation dysregulates normal cardiac function in zebrafish by a complex mechanism that involves alterations in both CaM-Ca2+ and CaM-RyR2 interactions. Herein, to gain further insight into how the CaM E105A mutation leads to severe cardiac arrhythmia, we generated large quantities of recombinant CaMWT and CaME105A proteins. We then performed ITC experiments to investigate and compare the interactions of CaMWT and CaME105A mutant protein with two synthetic peptides that correspond to the two aforementioned human RyR2 regions, which we have proposed to contribute to the RyR2 CaM-binding pocket. Our data reveal that the E105A mutation has a significant negative effect on the interaction of CaM with both RyR2 regions in the presence and absence of Ca2+, highlighting the potential contribution of these two human RyR2 regions to an RyR2 CaM-binding pocket, which may be essential for physiological CaM/RyR2 association and thus channel regulation.

[Study on 4 cases of mushroom poisoning with amanita and identification of poison].

Different kinds of poisonous mushrooms contain different toxic components. Acute liver injury caused by amanita mushroom is the main cause of death from poisonous mushroom poisoning in China. Consumption of poisonous mushrooms has an incubation period, there is a false recovery period in the clinical process, and the early performance is slight and does not attract enough attention from doctors, and it is easy to miss the treatment opportunity. The clinical characteristics, treatment and identification of mushrooms containing amanita in 4 patients were analyzed in order to improve clinicians' understanding of the diagnosis and treatment of mushroom poisoning and early species identification.

SPERM FACTORS AND EGG ACTIVATION: The structure and function relationship of sperm PLCZ1.

In 2002, sperm-specific phospholipase C zeta1 (PLCZ1) was discovered and through these 20 years, it has been established as the predominant sperm oocyte-activating factor. PLCZ1 cRNA expression or direct protein microinjection into mammalian oocytes triggers calcium (Ca2+) oscillations indistinguishable from those observed at fertilization. The imperative role of PLCZ1 in oocyte activation is revealed by the vast number of human mutations throughout the PLCZ1 gene that have been identified and directly linked with certain forms of male infertility due to oocyte activation deficiency. PLCZ1 is the smallest PLC in size, comprising four N-terminal EF-hand domains, followed by X and Y catalytic domains, which are separated by the XY-linker, and ending with a C-terminal C2 domain. The EF hands are responsible for the high Ca2+ sensitivity of PLCZ1. The X and Y catalytic domains are responsible for the catalysis of the phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] substrate to produce the Ca2+-mobilising messenger, inositol 1,4,5-trisphosphate (IP3), while the XY-linker plays multiple roles in the unique mode of PLCZ1 action. Finally, the C2 domain has been proposed to facilitate the anchoring of PLCZ1 to intracellular vesicles through its direct interactions with specific phosphoinositides. This review discusses recent advances in the structure and function relationship of PLCZ1 and the potential binding partners of this important sperm-specific protein in the sperm and oocyte. The unravelling of all the remaining hidden secrets of sperm PLCZ1 should help us to understand the precise mechanism of fertilization, as well as enabling the diagnosis and treatment of currently unknown forms of PLCZ1 -linked human infertility.

Economic burden of public health care and hospitalisation associated with COVID-19 in China.

OBJECTIVES: This study aimed to evaluate the socio-economic burden imposed on the Chinese healthcare system during the coronavirus disease 2019 (COVID-19) pandemic. STUDY DESIGN: A cross-sectional study was used to investigate how COVID-19 impacted health and medical costs in China. Data were derived from a subdivision of the Centers for Disease control and Prevention of China. METHODS: We prospectively collected information from the Centers for Disease Control and Prevention and the designated hospitals to determine the cost of public health care and hospitalisation due to COVID-19. We estimated the resource use and direct medical costs associated with public health. RESULTS: The average costs, per case, for specimen collection and nucleic acid testing (NAT [specifically, polymerase chain reaction {PCR}]) in low-risk populations were $29.49 and $53.44, respectively; however, the average cost of NAT in high-risk populations was $297.94 per capita. The average costs per 1000 population for epidemiological surveys, disinfectant, health education and centralised isolation were $49.54, $247.01, $90.22 and $543.72, respectively. A single hospitalisation for COVID-19 in China cost a median of $2158.06 ($1961.13-$2325.65) in direct medical costs incurred only during hospitalisation, whereas the total costs associated with hospitalisation of patients with COVID-19 were estimated to have reached nearly $373.20 million in China as of 20, May, 2020. The cost of public health care associated with COVID-19 as of 20, May, 2020 ($6.83 billion) was 18.31 times that of hospitalisation. CONCLUSIONS: This study highlights the magnitude of resources needed to treat patients with COVID-19 and control the COVID-19 pandemic. Public health measures implemented by the Chinese government have been valuable in reducing the infection rate and may be cost-effective ways to control emerging infectious diseases.

Antibiotics as a silent driver of climate change? A case study investigating methane production in freshwater sediments.

Methane (CH4) is the second most important greenhouse gas after carbon dioxide (CO2) and is inter alia produced in natural freshwater ecosystems. Given the rise in CH4 emissions from natural sources, researchers are investigating environmental factors and climate change feedbacks to explain this increment. Despite being omnipresent in freshwaters, knowledge on the influence of chemical stressors of anthropogenic origin (e.g., antibiotics) on methanogenesis is lacking. To address this knowledge gap, we incubated freshwater sediment under anaerobic conditions with a mixture of five antibiotics at four levels (from 0 to 5000 µg/L) for 42 days. Weekly measurements of CH4 and CO2 in the headspace, as well as their compound-specific δ13C, showed that the CH4 production rate was increased by up to 94% at 5000 µg/L and up to 29% at field-relevant concentrations (i.e., 50 µg/L). Metabarcoding of the archaeal and eubacterial 16S rRNA gene showed that effects of antibiotics on bacterial community level (i.e., species composition) may partially explain the observed differences in CH4 production rates. Despite the complications of transferring experimental CH4 production rates to realistic field conditions, the study indicated that chemical stressors contribute to the emissions of greenhouse gases by affecting the methanogenesis in freshwaters.

Association of cardiac myosin-binding protein-C with the ryanodine receptor channel - putative retrograde regulation?

The cardiac muscle ryanodine receptor-Ca2+ release channel (RyR2) constitutes the sarcoplasmic reticulum (SR) Ca2+ efflux mechanism that initiates myocyte contraction, while cardiac myosin-binding protein-C (cMyBP-C; also known as MYBPC3) mediates regulation of acto-myosin cross-bridge cycling. In this paper, we provide the first evidence for the presence of direct interaction between these two proteins, forming a RyR2-cMyBP-C complex. The C-terminus of cMyBP-C binds with the RyR2 N-terminus in mammalian cells and the interaction is not mediated by a fibronectin-like domain. Notably, we detected complex formation between both recombinant cMyBP-C and RyR2, as well as between the native proteins in cardiac tissue. Cellular Ca2+ dynamics in HEK293 cells is altered upon co-expression of cMyBP-C and RyR2, with lowered frequency of RyR2-mediated spontaneous Ca2+ oscillations, suggesting that cMyBP-C exerts a potential inhibitory effect on RyR2-dependent Ca2+ release. Discovery of a functional RyR2 association with cMyBP-C provides direct evidence for a putative mechanistic link between cytosolic soluble cMyBP-C and SR-mediated Ca2+ release, via RyR2. Importantly, this interaction may have clinical relevance to the observed cMyBP-C and RyR2 dysfunction in cardiac pathologies, such as hypertrophic cardiomyopathy.

Meta-analysis of the influence of lifestyle changes for preoperative weight loss on surgical outcomes.

BACKGROUND: The aim was to investigate whether preoperative weight loss results in improved clinical outcomes in surgical patients with clinically significant obesity. METHODS: This was a systematic review and aggregate data meta-analysis of RCTs and cohort studies. PubMed, MEDLINE, Embase and CINAHL Plus databases were searched from inception to February 2018. Eligibility criteria were: studies assessing the effect of weight loss interventions (low-energy diets with or without an exercise component) on clinical outcomes in patients undergoing any surgical procedure. Data on 30-day or all-cause in-hospital mortality were extracted and synthesized in meta-analyses. Postoperative thromboembolic complications, duration of surgery, infection and duration of hospital stay were also assessed. RESULTS: A total of 6060 patients in four RCTs and 12 cohort studies, all from European and North American centres, were identified. Most were in the field of bariatric surgery and all had some methodological limitations. The pooled effect estimate suggested that preoperative weight loss programmes were effective, leading to significant weight reduction compared with controls: mean difference -7·42 (95 per cent c.i. -10·09 to -4·74) kg (P < 0·001). Preoperative weight loss interventions were not associated with a reduction in perioperative mortality (odds ratio 1·41, 95 per cent c.i. 0·24 to 8·40; I2 = 0 per cent, P = 0·66) but the event rate was low. The weight loss groups had shorter hospital stay (by 27 per cent). No differences were found for morbidity. CONCLUSION: This limited preoperative weight loss has advantages but may not alter the postoperative morbidity or mortality risk.

Hypertrophic cardiomyopathy-linked variants of cardiac myosin-binding protein C3 display altered molecular properties and actin interaction.

The most common inherited cardiac disorder, hypertrophic cardiomyopathy (HCM), is characterized by thickening of heart muscle, for which genetic mutations in cardiac myosin-binding protein C3 (c-MYBPC3) gene, is the leading cause. Notably, patients with HCM display a heterogeneous clinical presentation, onset and prognosis. Thus, delineating the molecular mechanisms that explain how disparate c-MYBPC3 variants lead to HCM is essential for correlating the impact of specific genotypes on clinical severity. Herein, five c-MYBPC3 missense variants clinically associated with HCM were investigated; namely V1 (R177H), V2 (A216T), V3 (E258K), V4 (E441K) and double mutation V5 (V3 + V4), all located within the C1 and C2 domains of MyBP-C, a region known to interact with sarcomeric protein, actin. Injection of the variant complementary RNAs in zebrafish embryos was observed to recapitulate phenotypic aspects of HCM in patients. Interestingly, V3- and V5-cRNA injection produced the most severe zebrafish cardiac phenotype, exhibiting increased diastolic/systolic myocardial thickness and significantly reduced heart rate compared with control zebrafish. Molecular analysis of recombinant C0-C2 protein fragments revealed that c-MYBPC3 variants alter the C0-C2 domain secondary structure, thermodynamic stability and importantly, result in a reduced binding affinity to cardiac actin. V5 (double mutant), displayed the greatest protein instability with concomitant loss of actin-binding function. Our study provides specific mechanistic insight into how c-MYBPC3 pathogenic variants alter both functional and structural characteristics of C0-C2 domains leading to impaired actin interaction and reduced contractility, which may provide a basis for elucidating the disease mechanism in HCM patients with c-MYBPC3 mutations.

Calsequestrin interacts directly with the cardiac ryanodine receptor luminal domain.

Cardiac muscle contraction requires sarcoplasmic reticulum (SR) Ca2+ release mediated by the quaternary complex comprising the ryanodine receptor 2 (RyR2), calsequestrin 2 (CSQ2), junctin (encoded by ASPH) and triadin. Here, we demonstrate that a direct interaction exists between RyR2 and CSQ2. Topologically, CSQ2 binding occurs at the first luminal loop of RyR2. Co-expression of RyR2 and CSQ2 in a human cell line devoid of the other quaternary complex proteins results in altered Ca2+-release dynamics compared to cells expressing RyR2 only. These findings provide a new perspective for understanding the SR luminal Ca2+ sensor and its involvement in cardiac physiology and disease.

Cost-effectiveness of osteoporosis screening strategies for hip fracture prevention in older Chinese people: a decision tree modeling study in the Mr. OS and Ms. OS cohort in Hong Kong.

Despite the high costs of hip fracture, many governments provide limited support for osteoporosis screening. We demonstrated that osteoporosis screening by dual-energy X-ray absorptiometry (DXA) with or without pre-screening by Fracture Risk Assessment Tool (FRAX) or calcaneal ultrasound are more cost-effective than no screening in Chinese people aged 65 or over in Hong Kong. INTRODUCTION: To examine the cost-effective potential osteoporosis screening strategies for hip fracture prevention in Hong Kong. METHODS: Decision tree models were constructed to evaluate the cost per quality-adjusted life years (QALYs) of the different osteoporosis screening strategies followed by subsequent 5-year treatment with alendronate compared to no screening (but treat if a hip fracture occurs). The multiple osteoporosis screening strategies were composed of alternative tests and initiation age groups were evaluated with a 10-year horizon, and treatment were assigned if central dual-energy X-ray absorptiometry (DXA) T-score (at either the hip or spine) is - 2.5 or less. Strategies included DXA for all people and pre-screening with the Fracture Risk Assessment Tool (FRAX) at specific thresholds or by calcaneal quantitative ultrasonography (QUS) before taking DXA examination. All the model inputs were based on the Mr. OS and Ms. OS Hong Kong cohort; data are obtained from the Social Welfare Department or the published literature. RESULTS: All of the screening strategies, including the universal screening with DXA and the pre-screening with FRAX or QUS before DXA, were consistently more cost-effective than no screening for people aged 65 years old or over. One-way sensitivity analysis with a more optimistic assumption on treatment adherence or inclusion of other major osteoporotic fractures did not change the results materially. Probabilistic sensitivity analyses showed a dominant role of pre-screening with FRAX followed by subsequent osteoporosis drug treatment in people aged 70 years old or over in Hong Kong. CONCLUSIONS: Osteoporosis screening strategies based on DXA with or without pre-screening are more cost-effective compared to no screening for Chinese people aged 65 or over in Hong Kong.

Expression of sperm PLCζ and clinical outcomes of ICSI-AOA in men affected by globozoospermia due to DPY19L2 deletion.

Globozoospermia is characterized by the presence of 100% acrosomeless round-headed spermatozoa in an ejaculate. Failed fertilization after intracytoplasmic sperm injection (ICSI) is commonly reported for globozoospermic couples and can be overcome by artificial oocyte activation (AOA). Phospholipase C zeta (PLCζ) is one of the main sperm factors involved in oocyte activation and its low expression levels mainly account for fertilization failure. Deletion of the DPY19L2 gene is reported as a main genetic cause in over 70% of infertile men with globozoospermia. The current study assesses the expression profile of sperm PLCζ at RNA and protein levels in 32 DPY19L2 deletion-mediated globozoospermic men and reports corresponding clinical outcomes following ICSI with AOA. The expression of PLCζ relative to GAPDH at RNA (0.78 ± 0.16 versus 1.65 ± 0.24; P = 0.02) and protein (0.39 ± 0.12 versus 0.83 ± 0.13; P = 0.01) levels in globozoospermic men with DPY19L2 deletion was significantly lower compared with fertile men (n = 32). Fertilization rate in globozoospermic couples following ICSI-AOA was significantly lower compared with fertile men (53.14 ± 5.13% versus 87.64 ± 2.38%, P < 0.001). However, implantation (26.2%) and pregnancy (53.8%) rates were not jeopardized by DPY19L2 deletion in these couples.

A novel splice site mutation of the PRKAR1A gene, C.440+5 G>C, in a Chinese family with Carney complex.

BACKGROUND: Carney complex (CNC) is an extremely rare, multiple endocrine neoplasia syndrome that occurs in an autosomal dominant manner. Mutations in PRKAR1A have been reported to be a common genetic cause of CNC. METHODS: In this study, we reported a Chinese pedigree of CNC that manifests mainly as spotty skin pigmentation and primary pigmented nodular adrenocortical disease. Whole blood samples of this pedigree were collected for DNA/RNA analysis. Polymerase chain reaction (PCR) and reverse-transcription polymerase chain reaction analyses were performed to amplify the 11 exons and adjacent introns of PRKAR1A. Direct sequencing was used to detect the mutation, and DNA from 70 Han Chinese people was extracted and sequenced as a control to estimate the frequency of the identified mutation. RESULTS: Within the pedigree, ten patients with CNC were identified, and a novel heterozygous mutation (c.440+5 G>C in intron 4a) was identified in the PRKAR1A gene. PCR amplification of cDNA from the control subjects and patients was performed. Agarose gel electrophoresis showed only one wild-type band in the cDNA corresponding to the former group, whereas an extra band was present in samples from the latter group corresponding to the skipping of exon 4a; this confirms that the variant affects PRKAR1A splicing. CONCLUSION: In conclusion, the c.440+5 G>C mutation is a new splice site mutation that has not been reported and has the potential to broaden the mutational spectrum of PRKAR1A that is associated with CNC, which would facilitate genetic diagnosis and counseling for CNC.

The role and mechanism of action of sperm PLC-zeta in mammalian fertilisation.

At mammalian fertilisation, the fundamental stimulus that triggers oocyte (egg) activation and initiation of early embryonic development is an acute rise of the intracellular-free calcium (Ca2+) concentration inside the egg cytoplasm. This essential Ca2+ increase comprises a characteristic series of repetitive Ca2+ oscillations, starting soon after sperm-egg fusion. Over the last 15 years, accumulating scientific and clinical evidence supports the notion that the physiological stimulus that precedes the cytosolic Ca2+ oscillations is a novel, testis-specific phospholipase C (PLC) isoform, known as PLC-zeta (PLCζ). Sperm PLCζ catalyses the hydrolysis of phosphatidylinositol 4,5-bisphosphate triggering cytosolic Ca2+ oscillations through the inositol 1,4,5-trisphosphate signalling pathway. PLCζ is the smallest known mammalian PLC isoform with the most elementary domain organisation. However, relative to somatic PLCs, the PLCζ isoform possesses a unique potency in stimulating Ca2+ oscillations in eggs that is attributed to its novel biochemical characteristics. In this review, we discuss the latest developments that have begun to unravel the vital role of PLCζ at mammalian fertilisation and decipher its unique mechanism of action within the fertilising egg. We also postulate the significant potential diagnostic and therapeutic capacity of PLCζ in alleviating certain types of male infertility.

Male infertility-linked point mutation reveals a vital binding role for the C2 domain of sperm PLCζ.

Sperm-specific phospholipase C zeta (PLCζ) is widely considered to be the physiological stimulus that evokes intracellular calcium (Ca2+) oscillations that are essential for the initiation of egg activation during mammalian fertilisation. A recent genetic study reported a male infertility case that was directly associated with a point mutation in the PLCζ C2 domain, where an isoleucine residue had been substituted with a phenylalanine (I489F). Here, we have analysed the effect of this mutation on the in vivo Ca2+ oscillation-inducing activity and the in vitro biochemical properties of human PLCζ. Microinjection of cRNA or recombinant protein corresponding to PLCζI489F mutant at physiological concentrations completely failed to cause Ca2+ oscillations and trigger development. However, this infertile phenotype could be effectively rescued by microinjection of relatively high (non-physiological) amounts of recombinant mutant PLCζI489F protein, leading to Ca2+ oscillations and egg activation. Our in vitro biochemical analysis suggested that the PLCζI489F mutant displayed similar enzymatic properties, but dramatically reduced binding to PI(3)P and PI(5)P-containing liposomes compared with wild-type PLCζ. Our findings highlight the importance of PLCζ at fertilisation and the vital role of the C2 domain in PLCζ function, possibly due to its novel binding characteristics.