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Whole-exome sequencing reveals that a rare variant of phospholipase D3 ( PLD3 ( V232M )) segregates with Alzheimer’s disease status in two independent families and doubles risk for the disease in case–control series, and that several other PLD3 variants increase risk for Alzheimer’s disease in African Americans and people of European descent. New genetic risk variant for Alzheimer's disease The identification of mutations causing Alzheimer's disease in amyloid-β precursor protein, presenilin 1 and presenilin 2 led to a better understanding of the pathobiology of the condition. Further mutations are expected to be implicated, but the identification of such variants has been challenging. These authors used exome sequencing to identify low-frequency coding variants with large effects on late-onset Alzheimer's disease. They report several coding variants in the gene PLD3 , coding for phospholipase D3, that increase disease risk at least twofold. PLD3 may have a role in the processing of amyloid-β and may have potential as a novel therapeutic target. Genome-wide association studies (GWAS) have identified several risk variants for late-onset Alzheimer's disease (LOAD) 1 , 2 . These common variants have replicable but small effects on LOAD risk and generally do not have obvious functional effects. Low-frequency coding variants, not detected by GWAS, are predicted to include functional variants with larger effects on risk. To identify low-frequency coding variants with large effects on LOAD risk, we carried out whole-exome sequencing (WES) in 14 large LOAD families and follow-up analyses of the candidate variants in several large LOAD case–control data sets. A rare variant in PLD3 (phospholipase D3; Val232Met) segregated with disease status in two independent families and doubled risk for Alzheimer’s disease in seven independent case–control series with a total of more than 11,000 cases and controls of European descent. Gene-based burden analyses in 4,387 cases and controls of European descent and 302 African American cases and controls, with complete sequence data for PLD3 , reveal that several variants in this gene increase risk for Alzheimer’s disease in both populations. PLD3 is highly expressed in brain regions that are vulnerable to Alzheimer’s disease pathology, including hippocampus and cortex, and is expressed at significantly lower levels in neurons from Alzheimer’s disease brains compared to control brains. Overexpression of PLD3 leads to a significant decrease in intracellular amyloid-β precursor protein (APP) and extracellular Aβ42 and Aβ40 (the 42- and 40-residue isoforms of the amyloid-β peptide), and knockdown of PLD3 leads to a significant increase in extracellular Aβ42 and Aβ40. Together, our genetic and functional data indicate that carriers of PLD3 coding variants have a twofold increased risk for LOAD and that PLD3 influences APP processing. This study provides an example of how densely affected families may help to identify rare variants with large effects on risk for disease or other complex traits.

Genome-wide association studies (GWAS) have identified several risk variants for late-onset Alzheimer's disease (LOAD)1,2. These common variants have replicable but small effects on LOAD risk and generally do not have obvious functional effects. Low-frequency coding variants, not detected by GWAS, are predicted to include functional variants with larger effects on risk. To identify low frequency coding variants with large effects on LOAD risk, we performed whole exome-sequencing (WES) in 14 large LOAD families and follow-up analyses of the candidate variants in several large case-control datasets. A rare variant in PLD3 (phospholipase-D family, member 3, rs145999145; V232M) segregated with disease status in two independent families and doubled risk for AD in seven independent case-control series (V232M meta-analysis; OR= 2.10, CI=1.47-2.99; p= 2.93×10-5, 11,354 cases and controls of European-descent). Gene-based burden analyses in 4,387 cases and controls of European-descent and 302 African American cases and controls, with complete sequence data for PLD3, indicate that several variants in this gene increase risk for AD in both populations (EA: OR= 2.75, CI=2.05-3.68; p=1.44×10-11, AA: OR= 5.48, CI=1.77-16.92; p=1.40×10-3). PLD3 is highly expressed in brain regions vulnerable to AD pathology, including hippocampus and cortex, and is expressed at lower levels in neurons from AD brains compared to control brains (p=8.10×10-10). Over-expression of PLD3 leads to a significant decrease in intracellular APP and extracellular Aβ42 and Aβ40, while knock-down of PLD3 leads to a significant increase in extracellular Aβ42 and Aβ40. Together, our genetic and functional data indicate that carriers of PLD3 coding variants have a two-fold increased risk for LOAD and that PLD3 influences APP processing. This study provides an example of how densely affected families may be used to identify rare variants with large effects on risk for disease or other complex traits.

One of the driving structural requirements of the Habitable Exo-Planet (HabEx) telescope is to maintain Line Of Sight (LOS) stability between the Primary Mirror (PM) and Secondary Mirror (SM) of ≤ 5 mas. Dynamic analyses of two configurations of a proposed (HabEx) 4 meter off-axis telescope structure were performed to predict effects of jitter on primary/secondary mirror alignment. The dynamic disturbance used as the forcing function was the James Webb Space Telescope reaction wheel assembly vibration emission specification level. The objective of these analyses was to predict \"order-of-magnitude\" performance for various structural configurations which will roll into efforts to define the HabEx structural design's global architecture. Two variations of the basic architectural design were analyzed. Relative motion between the PM and the SM for each design configuration are reported.

The Skype-killing-speakers problem is very simple to...

To date, the use of traditional nucleic acid amplification tests (NAAT) for detection of HIV-1 DNA or RNA has been restricted to laboratory settings due to time, equipment, and technical expertise requirements. The availability of a rapid NAAT with applicability for resource-limited or point-of-care (POC) settings would fill a great need in HIV diagnostics, allowing for timely diagnosis or confirmation of infection status, as well as facilitating the diagnosis of acute infection, screening and evaluation of infants born to HIV-infected mothers. Isothermal amplification methods, such as reverse-transcription, loop-mediated isothermal amplification (RT-LAMP), exhibit characteristics that are ideal for POC settings, since they are typically quicker, easier to perform, and allow for integration into low-tech, portable heating devices. In this study, we evaluated the HIV-1 RT-LAMP assay using portable, non-instrumented nucleic acid amplification (NINA) heating devices that generate heat from the exothermic reaction of calcium oxide and water. The NINA heating devices exhibited stable temperatures throughout the amplification reaction and consistent amplification results between three separate devices and a thermalcycler. The performance of the NINA heaters was validated using whole blood specimens from HIV-1 infected patients. The RT-LAMP isothermal amplification method used in conjunction with a chemical heating device provides a portable, rapid and robust NAAT platform that has the potential to facilitate HIV-1 testing in resource-limited settings and POC.

Background The iPSC Neurodegenerative Disease Initiative (iNDI) is the largest‐ever induced pluripotent stem cell (iPSC) genome engineering project, modeling over 100 ADRD mutations in high‐quality isogenic human iPSCs. iNDI leverages unbiased CRISPRi screens as a powerful tool to identify fundamental mechanisms and modifiers of disease. However, current CRISPRi molecular tools are poorly optimized for use in iPSC‐derived neurons (iNeurons). Here we develop a Cre‐lox inducible CRISPRi system (CRISPRi‐Cre), enabling gene knockdown upon Cre delivery to postmitotic iNeurons, and identification of neuron‐specific, disease‐relevant modifiers. Method We modified a plasmid carrying a potent Zim3‐dCas9 transcriptional repressor to include a strong floxed STOP cassette upstream of the Zim3 start codon. We leveraged HaloTag‐TDP43 and HaloTag‐FUS iSPCs from the iNDI project paired with flow cytometry to validate leakiness and responsiveness to Cre in iPSCs and iNeurons treated with sgRNAs. We then performed a genome‐wide CRISPRi survival screen in iNeurons to demonstrate broad functionality of this inducible CRISPRi system with over 20,000 sgRNAs. Finally, we use CRISPRi‐Cre to identify neuron‐specific regulators of neuronal activity in iNeurons. Result We demonstrate that in the absence of Cre, dCas9 is inactive. Delivery of lentivirus‐Cre to iNeurons activates dCas9, resulting in potent gene knockdown. In genome‐wide CRISPRi screens, we show that CRISPRi‐Cre identifies many of the same hits observed in screens using constitutive‐active dCas9, and importantly uncovers novel neuron‐specific hits not identified in previous CRISPRi screens. Conclusion Here, we developed a robust Cre‐inducible CRISPRi system that enables post‐mitotic gene knockdown in iPSC‐derived neurons. Our CRISPRi screens identify neuron‐specific hits, demonstrating the utility of our tool to help uncover disease‐relevant mechanisms, modifiers, and potential therapeutic targets in relevant cell types.

Limited Proteolysis by Mass Spectrometry (LiP-MS) is a technique used to observe the structural changes of proteins on a proteome-wide scale. Limited proteolysis consists of a specific, or limited, number of proteases performing hydrolyzation on a large portion of proteins. This causes the protease to detect the weak portion of the hydrolyzed protein and cut it out. The technique of limited proteolysis coupled mass spectrometry aims to alleviate the limitations of classic proteomic analysis, such as being unable to detect protein structural alterations on a wide scale and at high throughput. In this experiment, we used iPSC-derived neurons treated with beta-amyloid and performed classic proteomics and LiP-MS in parallel to observe the proteome scale of structural changes caused by beta-amyloid. Through experimentation, it was found that the control group consisted of 180 protein profiles that the beta-amyloid treated group did not possess using the limited proteolysis-mass spectrometry technique. Additionally, various cleavage sites by proteinase K in the beta-amyloid treated neurons compared to the control neurons, as well as multiple molecular pathways, such as neuron projection cytoplasm, were identified.

The iPSC Neurodegenerative Disease Initiative (iNDI) is the largest-ever induced pluripotent stem cell (iPSC) genome engineering project, modeling over 100 ADRD mutations in high-quality isogenic human iPSCs. iNDI leverages unbiased CRISPRi screens as a powerful tool to identify fundamental mechanisms and modifiers of disease. However, current CRISPRi molecular tools are poorly optimized for use in iPSC-derived neurons (iNeurons). Here we develop a Cre-lox inducible CRISPRi system (CRISPRi-Cre), enabling gene knockdown upon Cre delivery to postmitotic iNeurons, and identification of neuron-specific, disease-relevant modifiers. We modified a plasmid carrying a potent Zim3-dCas9 transcriptional repressor to include a strong floxed STOP cassette upstream of the Zim3 start codon. We leveraged HaloTag-TDP43 and HaloTag-FUS iSPCs from the iNDI project paired with flow cytometry to validate leakiness and responsiveness to Cre in iPSCs and iNeurons treated with sgRNAs. We then performed a genome-wide CRISPRi survival screen in iNeurons to demonstrate broad functionality of this inducible CRISPRi system with over 20,000 sgRNAs. Finally, we use CRISPRi-Cre to identify neuron-specific regulators of neuronal activity in iNeurons. We demonstrate that in the absence of Cre, dCas9 is inactive. Delivery of lentivirus-Cre to iNeurons activates dCas9, resulting in potent gene knockdown. In genome-wide CRISPRi screens, we show that CRISPRi-Cre identifies many of the same hits observed in screens using constitutive-active dCas9, and importantly uncovers novel neuron-specific hits not identified in previous CRISPRi screens. Here, we developed a robust Cre-inducible CRISPRi system that enables post-mitotic gene knockdown in iPSC-derived neurons. Our CRISPRi screens identify neuron-specific hits, demonstrating the utility of our tool to help uncover disease-relevant mechanisms, modifiers, and potential therapeutic targets in relevant cell types.