Magnetic Nanoparticle-Assisted Non-Viral CRISPR-Cas9 for Enhanced Genome Editing to Treat Rett Syndrome.

The CRISPR-Cas9 technology has the potential to revolutionize the treatment of various diseases, including Rett syndrome, by enabling the correction of genes or mutations in human patient cells. However, several challenges need to be addressed before its widespread clinical application. These challenges include the low delivery efficiencies to target cells, the actual efficiency of the genome-editing process, and the precision with which the CRISPR-Cas system operates. Herein, the study presents a Magnetic Nanoparticle-Assisted Genome Editing (MAGE) platform, which significantly improves the transfection efficiency, biocompatibility, and genome-editing accuracy of CRISPR-Cas9 technology. To demonstrate the feasibility of the developed technology, MAGE is applied to correct the mutated MeCP2 gene in induced pluripotent stem cell-derived neural progenitor cells (iPSC-NPCs) from a Rett syndrome patient. By combining magnetofection and magnetic-activated cell sorting, MAGE achieves higher multi-plasmid delivery (99.3%) and repairing efficiencies (42.95%) with significantly shorter incubation times than conventional transfection agents without size limitations on plasmids. The repaired iPSC-NPCs showed similar characteristics as wild-type neurons when they differentiated into neurons, further validating MAGE and its potential for future clinical applications. In short, the developed nanobio-combined CRISPR-Cas9 technology offers the potential for various clinical applications, particularly in stem cell therapies targeting different genetic diseases.

Angiotensin Receptor Blockers and the Risk of Suspected Drug-Induced Liver Injury: A Retrospective Cohort Study Using Electronic Health Record-Based Common Data Model in South Korea.

INTRODUCTION: Angiotensin receptor blockers are widely used antihypertensive drugs in South Korea. In 2021, the Korea Ministry of Food and Drug Safety acknowledged the need for national compensation for a drug-induced liver injury (DILI) after azilsartan use. However, little is known regarding the association between angiotensin receptor blockers and DILI. OBJECTIVE: We conducted a retrospective cohort study in incident users of angiotensin receptor blockers from a common data model database (1 January, 2017-31 December, 2021) to compare the risk of DILI among specific angiotensin receptor blockers against valsartan. METHODS: Patients were assigned to treatment groups at cohort entry based on prescribed angiotensin receptor blockers. Drug-induced liver injury was operationally defined using the International DILI Expert Working Group criteria. Cox regression analyses were conducted to derive hazard ratios and the inverse probability of treatment weighting method was applied. All analyses were performed using R. RESULTS: In total, 229,881 angiotensin receptor blocker users from 20 university hospitals were included. Crude DILI incidence ranged from 15.6 to 82.8 per 1000 person-years in treatment groups, most were cholestatic and of mild severity. Overall, the risk of DILI was significantly lower in olmesartan users than in valsartan users (hazard ratio: 0.73 [95% confidence interval 0.55-0.96]). In monotherapy patients, the risk was significantly higher in azilsartan users than in valsartan users (hazard ratio: 6.55 [95% confidence interval 5.28-8.12]). CONCLUSIONS: We found a significantly higher risk of suspected DILI in patients receiving azilsartan monotherapy compared with valsartan monotherapy. Our findings emphasize the utility of real-world evidence in advancing our understanding of adverse drug reactions in clinical practice.

Aneurysm and Artery Dissection After Oral VEGFR-TKI Use in Adults With Cancer.

Importance: The association of tyrosine kinase inhibitors targeting vascular endothelial growth factor receptors (VEGFR-TKIs) with aneurysm and artery dissection (AAD) has been frequently reported in spontaneous reporting databases. Objective: To investigate the risk and incidence of AAD occurrence in patients with cancer treated with oral VEGFR-TKIs, with capecitabine as an active comparator. Design, Setting, and Participants: This national, historical cohort study was conducted using national claims data from the National Health Insurance Service in Korea from 2007 to 2020, with a 1-year follow-up. Patients with cancer aged 40 years or older prescribed oral VEGFR-TKIs or capecitabine were enrolled. Data were analyzed from September 2022 through April 2023. Exposure: Oral VEGFR-TKIs (sorafenib, regorafenib, vandetanib, sunitinib, lenvatinib, axitinib, and pazopanib) or capecitabine as a comparator. Main Outcomes and Measures: Hazard ratios (HRs) were used to investigate the association between VEGFR-TKI use and AAD after propensity score matching. The primary outcome was AAD, and secondary outcomes were aortic aneurysm and dissection and AAD with rupture. Outcomes were defined using International Statistical Classification of Diseases and Related Health Problems, Tenth Revision (ICD-10) diagnosis codes. Results: Among 127 710 patients with cancer eligible for the study (80 386 males [62.9%]; mean [SD] age, 62.6 [10.9] years), 37 308 patients received VEGFR-TKIs and 90 402 patients received capecitabine. Among 27 535 matched patients receiving VEGFR-TKIs, the incidence of AAD within 1 year of treatment initiation was 6.0 per 1000 person-years. The median (IQR) time to AAD onset in the matched AAD group was 114 (67-257) days after treatment initiation, with the highest incidence observed during the first 3 months (45 incidents vs 31, 17, and 16 incidents during 3- to 6-month, 6- to 9-month, and 9- to 12-month periods, respectively). Cox regression modeling showed that the risk of AAD occurrence was significantly higher among patients prescribed VEGFR-TKIs than those receiving capecitabine (HR, 1.48; 95% CI, 1.08-2.02); similar results were obtained among females (HR, 2.08; 95% CI, 1.26-3.42), older adults (aged ≥65 years; HR, 1.42; 95% CI, 1.01-1.99), and patients with dyslipidemia (HR, 1.58; 95% CI, 1.11-2.24). Conclusions and Relevance: In this study, the use of oral VEGFR-TKIs was associated with an increased risk of AAD occurrence. These findings elucidate vascular toxic effects and may provide a substantial reference for reducing the socioeconomic burden of adverse events associated with VEGFR-TKI use.

MicroRNA-593-5p contributes to cell death following exposure to 1-methyl-4-phenylpyridinium by targeting PTEN-induced putative kinase 1.

Neurodegenerative diseases are characterized by a decline in neuronal function and structure, leading to neuronal death. Understanding the molecular mechanisms of neuronal death is crucial for developing therapeutics. MiRs are small noncoding RNAs that regulate gene expression by degrading target mRNAs or inhibiting translation. MiR dysregulation has been linked to many neurodegenerative diseases, but the underlying mechanisms are not well understood. As mitochondrial dysfunction is one of the common molecular mechanisms leading to neuronal death in many neurodegenerative diseases, here we studied miRs that modulate neuronal death caused by 1-methyl-4-phenylpyridinium (MPP+), an inhibitor of complex I in mitochondria. We identified miR-593-5p, levels of which were increased in SH-SY5Y human neuronal cells, after exposure to MPP+. We found that intracellular Ca2+, but not of reactive oxygen species, mediated this miR-593-5p increase. Furthermore, we found the increase in miR-593-5p was due to enhanced stability, not increased transcription or miR processing. Importantly, we show the increase in miR-593-5p contributed to MPP+-induced cell death. Our data revealed that miR-593-5p inhibits a signaling pathway involving PTEN-induced putative kinase 1 (PINK1) and Parkin, two proteins responsible for the removal of damaged mitochondria from cells, by targeting the coding sequence of PINK1 mRNA. Our findings suggest that miR-593-5p contributes to neuronal death resulting from MPP+ toxicity, in part, by impeding the PINK1/Parkin-mediated pathway that facilitates the clearance of damaged mitochondria. Taken together, our observations highlight the potential significance of inhibiting miR-593-5p as a therapeutic approach for neurodegenerative diseases.

Narcotics information management system in South Korea: system development and innovation.

BACKGROUND: As the misuse and abuse of medical narcotics are increasing in South Korea, an information system for the integrated information management of medical narcotic drugs across the nation is needed. This paper presents the development process of the Narcotics Information Management System (NIMS) for the monitoring of medical narcotics usage and the results of its implementation. METHODS: As the NIMS enforces that all narcotics handlers digitally report all information on handling medical narcotic drugs, the functional requirements of the NIMS have been identified in accordance with the Narcotics Control Act. In addition to the functional requirements, the non-functional requirements of the NIMS have been elicited by major narcotics handlers and their associations. The non-functional requirements include privacy, availability, connectivity, interoperability, and data integrity. The system design with entity-relationship diagrams and its implementation processes have been presented. RESULTS: The NIMS encompasses all narcotic handlers, which comprise exporting, importing, and pharmaceutical companies; wholesalers; hospitals and clinics; and pharmacies, collecting over 120 million cases annually. It enables transparent monitoring throughout the life cycle, from manufacturing, sales, purchase, and disposal of narcotics. As a result, the number of prescriptions for medical narcotics has been reduced by 9.2%. CONCLUSIONS: To the best of our knowledge, the NIMS is the world's first system to manage all information on the total life cycle of medical narcotics, including imports, production, distribution, use, and disposal of drugs. This system has enabled the safety management and monitoring of medical narcotic drugs. Additionally, it provides consistent and transparent information to physicians and patients, leading to the autonomous safety management of narcotics. The successful development of the NIMS can provide guidelines for implementing a narcotics management system in other countries.

`Risk of cardiovascular disease associated with febuxostat versus allopurinol use in patients with gout: a retrospective cohort study in Korea.

Febuxostat is the drug used to treat hyperuricemia in patients with gout. Recently, the usage of Febuxostat has been controversial over the side effects in cardiovascular. The study aimed to comparatively analyze the risk of cardiovascular disease associated with febuxostat and allopurinol use in Korean patients with gout. A cohort study was conducted using national insurance claim data from the Health Insurance Review and Assessment Service (HIRA). Adult patients who were diagnosed with gout and prescribed febuxostat or allopurinol more than once from July 1, 2015, to June 30, 2018 were studied. The outcome was cardiovascular disease. Analysis was performed using Cox's proportional hazard model following 1:1 propensity score matching to estimate the hazard ratio with a 95% confidence interval. In total, 90,590 patients were defined as the final study cohort who had an average follow-up of 467 days, including 28,732 and 61,858 patients in the febuxostat and allopurinol groups, respectively. After the 1:1 propensity score matching, the risk of cardiovascular disease in the febuxostat group was significantly higher than in the allopurinol group (HR: 1.17; 95% CI: 1.10-1.24). In the sensitivity analysis, the risk of cardiovascular disease in the febuxostat group was significantly higher than in the allopurinol group (HR: 1.09; 95% CI: 1.04-1.15). However, further sensitivity analysis showed no statistically significant difference between the febuxostat group and allopurinol group after adjusting for cardiovascular disease history before the index date. Similarly, no statistically significant difference was found between the two drugs in the subgroup analysis. Febuxostat was not associated with a significantly increased risk of cardiovascular disease.

Analysis of the functional sequences in the promoter region of the human adhesion molecule close homolog of L1.

BACKGROUND: Close Homolog of L1 (CHL1) is a member of the L1 family of cell adhesion molecules. CHL1 gene is located on human chromosome 3 and has been linked to several pathologies, including 3p deletion syndrome, schizophrenia, and tumor growth and metastasis. OBJECTIVE: The goal of the present study was to determine which region of the CHL1 promoter is most competent in driving CHL1 gene expression. Methods: Five candidate DNA fragments in the promoter regions were selected by screening across six species for evolutionary conserved sequences. The activity of these five promoter regions was quantitatively evaluated using a GFP reporter gene in transfection experiments, performed in C6 glioma cells. RESULTS: Of the five promoter regions tested, three drove reporter GFP expression, with the conserved region 6 (CR6, Gene ID AC066595.5, 25851-26850) being the most active for transcription. CONCLUSION: The identification of the CR6 activity provides a better understanding of the regulatory mechanisms underlying CHL1 expression. It may help future discovery of therapeutic strategies that involve influencing critical promoter regions to drive transcriptional regulation of the mammalian CHL1 gene.HIGHLIGHTSConserved regions of CHL1 promoter sequences were identified by in-silico analysis.Five conserved regions were tested for gene regulatory activity using a reporter assay.Conserved regions CR5, CR6 and CR7 show gene regulatory function in a reporter assay.Co-transfection of CR5 and CR6 yielded the highest reporter activity.The core region of CR6 (CR6core) was identified as a cis-acting element.In-tandem promoter CR5core-CR6core was the best in a reporter assay.

MicroRNA-7 promotes motor function recovery following spinal cord injury in mice.

Spinal cord injury (SCI) is a devastating neurological condition for which there are no effective therapies. Following an initial injury, there is a cascade of multiple downstream events termed secondary injury. Thus, therapeutic approaches targeting a single pathway may not offer the best solution for treating SCI. One of the most attractive properties of microRNAs (miR) as potential therapeutics is that they are highly effective in regulating complex biological pathways by targeting multiple genes and pathways. The current study investigated the role of miR-7-5p (miR-7), which was previously shown to have neuroprotective functions, in promoting motor function recovery following SCI. We used an adeno-associated virus 1 (AAV1) vector to deliver the gene encoding miR-7 to the spinal cord of adult mice and found that this virus was mainly transduced into the neurons of the spinal cord. Transduction of AAV1-miR-7 improved hindlimb locomotor function following SCI over an 8-week observation period. This improvement was accompanied by reduced neuronal loss in the lesion. In addition, the beneficial effect of miR-7 was associated with enhanced levels of TH-positive axons in the lesion. Taken together, we suggest that miR-7 improves motor function recovery after SCI by protecting neuronal death and increasing axon levels. These findings suggest that miR-7 could be developed as a potential treatment for SCI in human.

The Necessity of LED to Ambient Light Ratio Optimization for Vehicular Optical Camera Communication.

In vehicular optical camera communication (VOCC) systems, LED panels are used to transmit visible light signals which are captured by cameras. The logic bits 1 and 0 are represented by the On and Off status of the LEDs in the panel. The bit error rate (BER) of the system is directly proportional to the distinguishability of the On and Off LEDs in the received LED panel images. The signal quality is commonly believed in telecommunications to improve with a higher transmitted power. Therefore, one might expect to get a lower BER in VOCC systems by simply using more powerful LED lights. However, this is not the case with VOCC systems. This paper shows that the LED distinguishability is simultaneously determined by two factors: The LED extinction ratio and LED interference. The former needs to be kept high and the latter kept low for better LED distinguishability. The problem is that both the extinction ratio and interference increase with the ratio of LED light to ambient light (L2A). Consequently, an optimal L2A ratio exists to achieve the optimal balance between the positive impact of the extinction ratio and the negative impact of the interference. This can bring about the lowest BER without changing the system's data rate. In addition, this paper shows that the optimal L2A ratio varies according to the interval between the LEDs in a panel. We analyze the effect of the L2A ratio and LED interval on LED distinguishability. We then formulate a constrained optimization problem to find the optimal L2A ratios corresponding to different LED intervals. The simulation results verify the necessity of LED to ambient light ratio optimization as it can bring about the lowest BER without scarifying other aspects of the VOCC system.

MicroRNA-7 facilitates the degradation of alpha-synuclein and its aggregates by promoting autophagy.

Alpha-Synuclein (α-Syn) is an important protein in the pathogenesis of Parkinson disease (PD) as it accumulates as fibrillar inclusions in affected brain regions including dopaminergic neurons in the substantia nigra. Elevated levels of α-Syn seem to be crucial in mediating its toxicity. Thus, detailed information regarding the regulatory mechanism of α-Syn expression in several layers such as transcription, post-transcription and post-translation is needed in order to devise therapeutic interventions for PD. Previously, we reported that expression of α-Syn is repressed by microRNA-7 (miR-7) through its effect on the 3'-untranslated region (UTR) of α-Syn mRNA. Here, we show that miR-7 also accelerates the clearance of α-Syn and its aggregates by promoting autophagy in differentiated ReNcell VM cells. Further, miR-7 facilitates the degradation of pre-formed fibrils of α-Syn transported from outside the cells. This additional mechanism for reducing α-Syn levels show miR-7 to be an important molecular target for PD and other alpha-synucleinopathies.

The L1 adhesion molecule normalizes neuritogenesis in Rett syndrome-derived neural precursor cells.

Therapeutic intervention is an important need in ameliorating the severe consequences of Rett Syndrome (RTT), a neurological disorder caused by mutations in the X-linked gene methyl-CpG-binding protein-2 (MeCP2). Following previously observed morphological defects in induced pluripotent stem cell (iPSC)-derived neurons obtained from female RTT patients, we hypothesized that transfection with the L1 cell adhesion molecule (L1) could contribute to normalizing a pathological male cell system bearing a nonsense mutation of MeCP2. We found a decreased expression of L1 in RTT iPSCs-derived neural precursor cells (RTT NPCs) and decreased neuritogenesis. Expression of wild-type MeCP2 in RTTNPCs revealed a positive correlation between the levels of MeCP2 and L1, and normalization of cell survival. Expression of L1 in RTTNPCs enhanced neuritogenesis and soma size. Knock-down of MeCP2 in wild type NPCs reduced neuritogenesis. L1 expression is regulated by the MeCP2 promoter. These results suggest that a deficiency in L1 may partially account for RTT phenotypes.

A Probability-Based Algorithm Using Image Sensors to Track the LED in a Vehicle Visible Light Communication System.

This paper proposes a probability-based algorithm to track the LED in vehicle visible light communication systems using a camera. In this system, the transmitters are the vehicles' front and rear LED lights. The receivers are high speed cameras that take a series of images of the LEDs. ThedataembeddedinthelightisextractedbyfirstdetectingthepositionoftheLEDsintheseimages. Traditionally, LEDs are detected according to pixel intensity. However, when the vehicle is moving, motion blur occurs in the LED images, making it difficult to detect the LEDs. Particularly at high speeds, some frames are blurred at a high degree, which makes it impossible to detect the LED as well as extract the information embedded in these frames. The proposed algorithm relies not only on the pixel intensity, but also on the optical flow of the LEDs and on statistical information obtained from previous frames. Based on this information, the conditional probability that a pixel belongs to a LED is calculated. Then, the position of LED is determined based on this probability. To verify the suitability of the proposed algorithm, simulations are conducted by considering the incidents that can happen in a real-world situation, including a change in the position of the LEDs at each frame, as well as motion blur due to the vehicle speed.

VLC-Based Positioning System for an Indoor Environment Using an Image Sensor and an Accelerometer Sensor.

Recently, it is believed that lighting and communication technologies are being replaced by high power LEDs, which are core parts of the visible light communication (VLC) system. In this paper, by taking advantages of VLC, we propose a novel design for an indoor positioning system using LEDs, an image sensor (IS) and an accelerometer sensor (AS) from mobile devices. The proposed algorithm, which provides a high precision indoor position, consists of four LEDs mounted on the ceiling transmitting their own three-dimensional (3D) world coordinates and an IS at an unknown position receiving and demodulating the signals. Based on the 3D world coordinates and the 2D image coordinate of LEDs, the position of the mobile device is determined. Compared to existing algorithms, the proposed algorithm only requires one IS. In addition, by using an AS, the mobile device is allowed to have arbitrary orientation. Last but not least, a mechanism for reducing the image sensor noise is proposed to further improve the accuracy of the positioning algorithm. A simulation is conducted to verify the performance of the proposed algorithm.

An in-Depth Survey of Visible Light Communication Based Positioning Systems.

While visible light communication (VLC) has become the candidate for the wireless technology of the 21st century due to its inherent advantages, VLC based positioning also has a great chance of becoming the standard approach to positioning. Within the last few years, many studies on VLC based positioning have been published, but there are not many survey works in this field. In this paper, an in-depth survey of VLC based positioning systems is provided. More than 100 papers ranging from pioneering papers to the state-of-the-art in the field were collected and classified based on the positioning algorithms, the types of receivers, and the multiplexing techniques. In addition, current issues and research trends in VLC based positioning are discussed.

Performance Analysis of Visible Light Communication Using CMOS Sensors.

This paper elucidates the fundamentals of visible light communication systems that use the rolling shutter mechanism of CMOS sensors. All related information involving different subjects, such as photometry, camera operation, photography and image processing, are studied in tandem to explain the system. Then, the system performance is analyzed with respect to signal quality and data rate. To this end, a measure of signal quality, the signal to interference plus noise ratio (SINR), is formulated. Finally, a simulation is conducted to verify the analysis.

Analysis of human embryonic stem cells with regulatable expression of the cell adhesion molecule l1 in regeneration after spinal cord injury.

Cell replacement therapy is one potential avenue for central nervous system (CNS) repair. However, transplanted stem cells may not contribute to long-term recovery of the damaged CNS unless they are engineered for functional advantage. To fine tune regenerative capabilities, we developed a human neural cell line expressing L1, a regeneration-conducive adhesion molecule, under the control of a doxycycline regulatable Tet-off promoter. Controlled expression of L1 is desired because overexpression after regenerative events may lead to adverse consequences. The regulated system was tested in several cell lines, where doxycycline completely eliminated green fluorescent protein or L1 expression by 3-5 days in vitro. Increased colony formation as well as decreased proliferation were observed in H9NSCs without doxycycline (hL1-on). To test the role of L1 in vivo after acute compression spinal cord injury of immunosuppressed mice, quantum dot labeled hL1-on or hL1-off cells were injected at three sites: lesion; proximal; and caudal. Mice transplanted with hL1-on cells showed a better Basso Mouse Scale score, when compared to those with hL1-off cells. As compared to the hL1-off versus hL1-on cell transplanted mice 6 weeks post-transplantation, expression levels of L1, migration of transplanted cells, and immunoreactivity for tyrosine hydroxylase were higher, whereas expression of chondroitin sulfate proteoglycans was lower. Results indicate that L1 expression is regulatable in human stem cells by doxycycline in a nonviral engineering approach. Regulatable expression in a prospective nonleaky Tet-off system could hold promise for therapy, based on the multifunctional roles of L1, including neuronal migration and survival, neuritogenesis, myelination, and synaptic plasticity.

Arylsulfatase B improves locomotor function after mouse spinal cord injury.

Bacterial chondroitinase ABC (ChaseABC) has been used to remove the inhibitory chondroitin sulfate chains from chondroitin sulfate proteoglycans to improve regeneration after rodent spinal cord injury. We hypothesized that the mammalian enzyme arylsulfatase B (ARSB) would also enhance recovery after mouse spinal cord injury. Application of the mammalian enzyme would be an attractive alternative to ChaseABC because of its more robust chemical stability and reduced immunogenicity. A one-time injection of human ARSB into injured mouse spinal cord eliminated immunoreactivity for chondroitin sulfates within five days, and up to 9 weeks after injury. After a moderate spinal cord injury, we observed improvements of locomotor recovery assessed by the Basso Mouse Scale (BMS) in ARSB treated mice, compared to the buffer-treated control group, at 6 weeks after injection. After a severe spinal cord injury, mice injected with equivalent units of ARSB or ChaseABC improved similarly and both groups achieved significantly more locomotor recovery than the buffer-treated control mice. Serotonin and tyrosine hydroxylase immunoreactive axons were more extensively present in mouse spinal cords treated with ARSB and ChaseABC, and the immunoreactive axons penetrated further beyond the injury site in ARSB or ChaseABC treated mice than in control mice. These results indicate that mammalian ARSB improves functional recovery after CNS injury. The structural/molecular mechanisms underlying the observed functional improvement remain to be elucidated.

Increased cellular turnover in response to fluoxetine in neuronal precursors derived from human embryonic stem cells.

Previous reports have shown that antidepressants increase neuronal cell proliferation and enhance neuroplasticity both in vivo and in vitro. This study investigated the direct effects of one such antidepressant, fluoxetine , on cell proliferation and on the production of neurotrophic factors in neuronal precursors derived from human embryonic stem cells (hESCs; H9). Fluoxetine induced the differentiation of neuronal precursors, strongly enhancing neuronal characteristics. The rate of proliferation was higher in fluoxetine -treated cells than in control cells, as determined by MTT [3(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide] assay. The CPDL (cumulative population doubling level) of the fluoxetine-treated cells was significantly increased in comparison to that of control cells (p<.001). Bromodeoxyuridine incorporation and staurosporine-induced apoptosis assays were elevated in fluoxetine-treated cells. Quantitative RT-PCR analysis revealed no significant differences in the expression of neurotrophic factors, brain-derived neurotrophic factor (BDNF);glial-derived neurotrophic factor (GDNF) and cAMP-responsive element-binding protein (CREB) between cells treated with fluoxetine for two weeks and their untreated counterparts. These results may help elucidate the mechanism of action of fluoxetine as a therapeutic drug for the treatment of depression. Data presented herein provide more evidence that, in addition to having a direct chemical effect on serotonin levels, fluoxetine can influence hESC-derived neuronal cells by increasing cell proliferation, while allowing them to maintain their neuronal characteristics.

Human sperm devoid of PLC, zeta 1 fail to induce Ca(2+) release and are unable to initiate the first step of embryo development.

Egg activation, which is the first step in the initiation of embryo development, involves both completion of meiosis and progression into mitotic cycles. In mammals, the fertilizing sperm delivers the activating signal, which consists of oscillations in free cytosolic Ca(2+) concentration ([Ca(2+)](i)). Intracytoplasmic sperm injection (ICSI) is a technique that in vitro fertilization clinics use to treat a myriad of male factor infertility cases. Importantly, some patients who repeatedly fail ICSI also fail to induce egg activation and are, therefore, sterile. Here, we have found that sperm from patients who repeatedly failed ICSI were unable to induce [Ca(2+)](i) oscillations in mouse eggs. We have also shown that PLC, zeta 1 (PLCZ1), the sperm protein thought to induce [Ca(2+)](i) oscillations, was localized to the equatorial region of wild-type sperm heads but was undetectable in sperm from patients who had failed ICSI. The absence of PLCZ1 in these patients was further confirmed by Western blot, although genomic sequencing failed to reveal conclusive PLCZ1 mutations. Using mouse eggs, we reproduced the failure of sperm from these patients to induce egg activation and rescued it by injection of mouse Plcz1 mRNA. Together, our results indicate that the inability of human sperm to initiate [Ca(2+)](i) oscillations leads to failure of egg activation and sterility and that abnormal PLCZ1 expression underlies this functional defect.