A homing rescue gene drive with multiplexed gRNAs reaches high frequency in cage populations but generates functional resistance.

CRISPR homing gene drives have considerable potential for managing populations of medically and agriculturally significant insects. They operate by Cas9 cleavage followed by homology-directed repair, copying the drive allele to the wild-type chromosome and thus increasing in frequency and spreading throughout a population. However, resistance alleles formed by end-joining repair pose a significant obstacle. To address this, we create a homing drive targeting the essential hairy gene in Drosophila melanogaster. Nonfunctional resistance alleles are recessive lethal, while drive carriers have a recoded "rescue" version of hairy. The drive inheritance rate is moderate, and multigenerational cage studies show drive spread to 96%-97% of the population. However, the drive does not reach 100% due to the formation of functional resistance alleles, despite using four gRNAs. These alleles have a large deletion but likely utilize an alternate start codon. Thus, revised designs targeting more essential regions of a gene may be necessary to avoid such functional resistance. Replacement of the rescue element's native 3' UTR with a homolog from another species increases drive inheritance by 13%-24%. This was possibly because of reduced homology between the rescue element and surrounding genomic DNA, which could also be an important design consideration for rescue gene drives.

Full-length Isoform Sequencing for Resolving the Molecular Basis of Charcot-Marie-Tooth 2A.

Objectives: Transcript sequencing of patient-derived samples has been shown to improve the diagnostic yield for solving cases of suspected Mendelian conditions, yet the added benefit of full-length long-read transcript sequencing is largely unexplored. Methods: We applied short-read and full-length transcript sequencing and mitochondrial functional studies to a patient-derived fibroblast cell line from an individual with neuropathy that previously lacked a molecular diagnosis. Results: We identified an intronic homozygous MFN2 c.600-31T>G variant that disrupts the branch point critical for intron 6 splicing. Full-length long-read isoform complementary DNA (cDNA) sequencing after treatment with a nonsense-mediated mRNA decay (NMD) inhibitor revealed that this variant creates 5 distinct altered splicing transcripts. All 5 altered splicing transcripts have disrupted open reading frames and are subject to NMD. Furthermore, a patient-derived fibroblast line demonstrated abnormal lipid droplet formation, consistent with MFN2 dysfunction. Although correctly spliced full-length MFN2 transcripts are still produced, this branch point variant results in deficient MFN2 levels and autosomal recessive Charcot-Marie-Tooth disease, axonal, type 2A (CMT2A). Discussion: This case highlights the utility of full-length isoform sequencing for characterizing the molecular mechanism of undiagnosed rare diseases and expands our understanding of the genetic basis for CMT2A.

Dominant-negative variant in SLC1A4 causes an autosomal dominant epilepsy syndrome.

SLC1A4 is a trimeric neutral amino acid transporter essential for shuttling L-serine from astrocytes into neurons. Individuals with biallelic variants in SLC1A4 are known to have spastic tetraplegia, thin corpus callosum, and progressive microcephaly (SPATCCM) syndrome, but individuals with heterozygous variants are not thought to have disease. We identify an 8-year-old patient with global developmental delay, spasticity, epilepsy, and microcephaly who has a de novo heterozygous three amino acid duplication in SLC1A4 (L86_M88dup). We demonstrate that L86_M88dup causes a dominant-negative N-glycosylation defect of SLC1A4, which in turn reduces the plasma membrane localization of SLC1A4 and the transport rate of SLC1A4 for L-serine.

Assessment of distant-site rescue elements for CRISPR toxin-antidote gene drives.

Gene drive is a genetic engineering technology that can enable super-mendelian inheritance of specific alleles, allowing them to spread through a population. New gene drive types have increased flexibility, offering options for confined modification or suppression of target populations. Among the most promising are CRISPR toxin-antidote gene drives, which disrupt essential wild-type genes by targeting them with Cas9/gRNA. This results in their removal, increasing the frequency of the drive. All these drives rely on having an effective rescue element, which consists of a recoded version of the target gene. This rescue element can be at the same site as the target gene, maximizing the chance of efficient rescue, or at a distant site, which allows useful options such as easily disrupting another essential gene or increasing confinement. Previously, we developed a homing rescue drive targeting a haplolethal gene and a toxin-antidote drive targeting a haplosufficient gene. These successful drives had functional rescue elements but suboptimal drive efficiency. Here, we attempted to construct toxin-antidote drives targeting these genes with a distant-site configuration from three loci in Drosophila melanogaster. We found that additional gRNAs increased cut rates to nearly 100%. However, all distant-site rescue elements failed for both target genes. Furthermore, one rescue element with a minimally recoded sequence was used as a template for homology-directed repair for the target gene on a different chromosomal arm, resulting in the formation of functional resistance alleles. Together, these results can inform the design of future CRISPR-based toxin-antidote gene drives.

Full-length isoform sequencing for resolving the molecular basis of Charcot-Marie-Tooth 2A.

Objectives: Transcript sequencing of patient derived samples has been shown to improve the diagnostic yield for solving cases of likely Mendelian disorders, yet the added benefit of full-length long-read transcript sequencing is largely unexplored. Methods: We applied short-read and full-length isoform cDNA sequencing and mitochondrial functional studies to a patient-derived fibroblast cell line from an individual with neuropathy that previously lacked a molecular diagnosis. Results: We identified an intronic homozygous MFN2 c.600-31T>G variant that disrupts a branch point critical for intron 6 spicing. Full-length long-read isoform cDNA sequencing after treatment with a nonsense-mediated mRNA decay (NMD) inhibitor revealed that this variant creates five distinct altered splicing transcripts. All five altered splicing transcripts have disrupted open reading frames and are subject to NMD. Furthermore, a patient-derived fibroblast line demonstrated abnormal lipid droplet formation, consistent with MFN2 dysfunction. Although correctly spliced full-length MFN2 transcripts are still produced, this branch point variant results in deficient MFN2 protein levels and autosomal recessive Charcot-Marie-Tooth disease, axonal, type 2A (CMT2A). Discussion: This case highlights the utility of full-length isoform sequencing for characterizing the molecular mechanism of undiagnosed rare diseases and expands our understanding of the genetic basis for CMT2A.

Recording of a holographic cylindrical vector beam converter with a truncated cone prism.

This Letter proposes a holographic cylindrical vector beam converter (HCVBC) design that incorporates a continuously polarization-selective volume hologram circular-grating. A specially designed truncated cone prism is adopted for recording, which is conducted with a single incident, expanded, radially polarized beam. A prototype of this HCVBC was recorded and tested successfully. This design has the advantages of high diffraction efficiency, a narrow band, compactness, and planar configuration; thus, it is especially suitable for low-cost mass production and has high potential for application in related fields.

A RE-AIM Framework Analysis of DNA-Based Population Screening: Using Implementation Science to Translate Research Into Practice in a Healthcare System.

Introduction: DNA-based population screening has been proposed as a public health solution to identify individuals at risk for serious health conditions who otherwise may not present for medical care. The clinical utility and public health impact of DNA-based population screening is a subject of active investigation. Geisinger, an integrated healthcare delivery system, was one of the first healthcare systems to implement DNA screening programs (MyCode Community Health Initiative (MyCode) and clinical DNA screening pilot) that leverage exome data to identify individuals at risk for developing conditions with potential clinical actionability. Here, we demonstrate the use of an implementation science framework, RE-AIM (Reach, Effectiveness, Adoption, Implementation and Maintenance), to conduct a post-hoc evaluation and report outcomes from these two programs to inform the potential impact of DNA-based population screening. Methods: Reach and Effectiveness outcomes were determined from the MyCode research program, while Adoption and Implementation outcomes were measured using the clinical DNA screening pilot. Reach was defined as the number of patients who were offered and consented to participate in MyCode. Effectiveness of DNA screening was measured by reviewing MyCode program publications and synthesizing findings from themes. Adoption was measured by the total number of DNA screening tests ordered by clinicians at the clinical pilot sites. Implementation was assessed by interviewing a subset of clinical pilot clinicians about the deployment of and recommended adaptations to the pilot that could inform future program dissemination. Results: Reach: As of August 2020, 68% (215,078/316,612) of individuals approached to participate in the MyCode program consented. Effectiveness: Published evidence reported from MyCode demonstrates that DNA screening identifies at-risk individuals more comprehensively than clinical ascertainment based on phenotypes or personal/family history. Adoption: From July 2018 to June 2021, a total of 1,026 clinical DNA screening tests were ordered by 60 clinicians across the three pilot clinic sites. Implementation: Interviews with 14 clinicians practicing at the pilot clinic sites revealed motivation to provide patients with DNA screening results and yielded future implementation strategies. Conclusion: The RE-AIM framework offers a pragmatic solution to organize, analyze, and report outcomes across differently resourced and designed precision health programs that include genomic sequencing and return of clinically actionable genomic information.

Measurement of the refractive index of lenses from the maximum phase difference of the total internal reflection with a polarization camera.

The proposed total internal reflection (TIR)-based technique can be used for measuring the refractive index of lenses. Distribution of the phase difference between the s- and p-polarization states of the reflected light induced by TIR can be obtained by a polarization camera. The refractive index of the lens can be determined from the detected maximum phase difference, with the specific measurement equation. Only the maximum phase difference needs to be measured. Information about the incident angle, thickness of the lens, and the matching liquid is not needed. The experimental results demonstrate that the resolution of the system can reach 4.8×10-4RIU.

High Response CO Sensor Based on a Polyaniline/SnO2 Nanocomposite.

A polyaniline (PANI)/tin oxide (SnO2) composite for a CO sensor was fabricated using a composite film composed of SnO2 nanoparticles and PANI deposition in the present study. Tin oxide nanoparticles were synthesized by the sol-gel method. The SnO2 nanoparticles provided a high surface area to significantly enhance the response to the change in CO concentration at low operating temperature (<75 °C). The excellent sensor response was mainly attributed to the relatively good properties of PANI in the redox reaction during sensing, which produced a great resistance difference between the air and CO gas at low operating temperature. Therefore, the combination of n-type SnO2 nanoparticles with a high surface area and a thick film of conductive PANI is an effective strategy to design a high-performance CO gas sensor.

Prism-hologram-prism sandwiched recording method for polarization-selective substrate-mode volume holograms with a large diffraction angle.

We propose a prism-hologram-prism sandwiched recording method for the fabrication of polarization-selective substrate-mode volume holograms with a large diffraction angle. In fabrication, the C-RT20 photopolymer is sandwiched between two 45°-90°-45° prisms and the interference fringes can be easily recorded in the recording material. The experimental results are in good agreement with the theoretical predictions. The proposed method features of a reflection-type recording setup for a transmission element and belongs to a technique of longer-wavelength construction for shorter-wavelength reconstruction. In addition, the method is much easier than the traditional recording method of two incident beam interference and has application potential in holographic photonics.

Heterodyne moiré surface profilometry.

In this study, a novel moiré fringe analysis technique is proposed for measuring the surface profile of an object. After applying a relative displacement between two gratings at a constant velocity, every pixel of CMOS camera can capture a heterodyne moiré signal. The precise phase distribution of the moiré fringes can be extracted using a one-dimensional fast Fourier transform (FFT) analysis on every pixel, simultaneously filtering the harmonic noise of the moiré fringes. Finally, the surface profile of the tested objected can be generated by substituting the phase distribution into the relevant equation. The findings demonstrate the feasibility of this measuring method, and the measurement error was approximately 4.3 µm. The proposed method exhibits the merits of the Talbot effect, projection moiré method, FFT analysis, and heterodyne interferometry.

Multiport optical circulator by using polarizing beam splitter cubes as spatial walk-off polarizers.

Optical circulators are necessary passive devices applied in optical communication systems. In the design of optical circulators, the implementation of the function of spatial walk-off polarizers is a key technique that significantly influences the performance and cost of a device. This paper proposes a design of a multiport optical circulator by using polarizing beam splitter cubes as spatial walk-off polarizers. To show the feasibility of the design, a prototype of a six-port optical circulator was fabricated. The insertion losses are 0.94-1.49 dB, the isolations are 25-51 dB, and return losses are 27.72 dB.

Measurement of the pretilt angle and the cell gap of nematic liquid crystal cells by heterodyne interferometry.

This work proposes a simple method, based on the crystal rotation technique and heterodyne interferometry, to simultaneously determine the pretilt angle and cell gap of nematic liquid crystal cells. When heterodyne light passes through a nematic liquid crystal cell, the phase retardation given by the characteristic parameters of the cell can be measured accurately by heterodyne interferometry. This phase retardation relates to the pretilt angle, cell gap, and angle of incidence on the cell. By using the measured phase retardations at two incident angles, the pretilt angle and cell gap of the nematic liquid crystal cell can be estimated by numerical analysis. This method is feasible, requiring only two incident angles and prior knowledge of two characteristic parameters--extraordinary and ordinary refractive indices of the liquid crystal. It is characterized by the advantages of simplicity of installation, ease of operation, high stability, high accuracy, and high resolution.

Optical method for measuring optical rotation angle and refractive index of chiral solution.

Based on the phenomena of Brewster's angle and the principles of common-path heterodyne interferometry, we present an optical method for measuring the optical rotation angle and the refractive index of a chiral solution simultaneously in one optical configuration. A heterodyne light beam and a circularly polarized heterodyne light beam are separately guided to project onto the interface of a semicircle glass and a chiral solution. One of the beams is transmitted through the solution, and the other is reflected near Brewster's angle at the interface. Then the two beams pass through polarization components respectively for interference. The phase differences of the two interference signals used to determine the rotation angle and the refractive index become very high with the proper azimuth angles of some polarization components, hence achieving an accurate rotational angle and a refractive index. The feasibility of the measuring method was demonstrated by our experimental results. This method should bear the merits of high accuracy, short sample medium length, and simpler operational endeavor.

[Determination of quercetin in apples by reversed-phase high performance liquid chromatography].

A method for the determination of quercetin in pulp and peel of apples by reversed-phase high performance liquid chromatography with internal standard was developed. Samples were frozen at -80 degrees C for 24 h, then added 6 mol/L hydrochloride under the protection of antioxidant 2,6-di-tert-butyl-p-cresol. The slurry was hydrolyzed thermostatically at 90 degrees C for 2 h and centrifugated for 10 min. The separation was performed on an ODS column (150 mm x 6.0 mm i.d., 5 microm). Methanol-water (55:45, v/v) (pH adjusted to 3.3 with acetic acid) was used as the mobile phase with a flow rate of 1.0 mL/min. The injection volume was 20 microL. The detection wavelength was 370 nm. The results showed that the quercetin content in pulp and in peel were 3.11-10.78 microg/g and 253.57-744.59 microg/g, respectively. The mean recovery of quercetin in apple pulp was 100.4%. The method is simple, accurate, and reliable.

Multi-port polarization-independent optical quasi-circulators by using a pair of holographic spatial- and polarization- modules.

In this paper we proposed an alternative type of multi-port polarization-independent optical quasi-circulator by using a pair of holographic spatial- and polarization- modules. The prototype is fabricated and experimentally tested. In addition, the operating principles, the characteristics and the performances of this device are discussed. The merits of this design include polarization-independence, compactness, high isolation, low polarization mode dispersion, and easy fabrication. Furthermore, the number of ports can be scaled up easily.

Improved N-port optical quasi-circulator by using a pair of orthogonal holographic spatial- and polarization- modules.

Based on the flexibilities of light beam propagation in three dimensions, we propose an improved N-port optical quasi-circulator by using a pair of orthogonal holographic spatial- and polarization- modules. All optical elements are located in parallel planes that are perpendicular to the optical axis. The number of optical elements is decreased, and a higher performance optical quasi-circulator without crosstalk and polarization mode dispersion can be easily achieved. A prototype of 5-port polarizationindependent optical quasi-circulator operating at a wavelength of 1300nm was assembled and tested to show its validities.

Holographic spatial walk-off polarizer and its application to a 4-port polarization-independent optical circulator.

Based on the coupled-wave theory, a holographic spatial walk-off polarizer (HSWP) is designed. This HSWP is a transmission-type phase volume holographic grating on a substrate and its optical recording geometry can be derived from Chen's corrected methodology with a desired reconstruction condition. A pair of fabricated HSWPs with the splitting angle of 60 degrees is applied to assemble a new type of 4-port polarizationindependent optical circulator. The operating principles and the characteristics of the proposed HSWP and the prototype optical circulator are discussed.

[Effects of fenvalerate on reproductive and endocrine systems of male rats].

OBJECTIVES: To evaluate the impairment of fenvalerate on reproductive and endocrine systems and clarify the mechanism of action. METHODS: Different doses of fenvalerate (0, 2, 4, 12, 60 mg/kg) were orally treated to the adult male SD rats for 15 days and 30 days, respectively. The levels of serum follicle stimulating hormone(FSH), luteinizing hormone (LH), testosterone(T) and testis homogenate T were determined by radioimmunoassay(RIA). Besides, the activity of testicular marked enzymes such as acid phosphatases(ACP) and gamma-glutamyl transpeptidase(gamma-GT) were examined, and sperm head counts were measured to explain the changes of daily sperm production(Spr). RESULTS: In fifteen days, serum FSH levels markedly increased in rats exposed to fenvalerate of < or = 12 mg/kg groups(P < 0.01) and serum levels of LH increased in 12 mg/kg group(P < 0.01). In addition, T levels in testis homogenates decreased after treated with the doses of > or = 12 mg/kg groups compared with the control group(P < 0.01). In thirty days, serum contents of FSH were significantly elevated in the doses of > or = 12 mg/kg groups(P < 0.01) and homogenate levels of T were diminished in the low dose group(2.4 mg/kg) (P < 0.05). Activity of ACP increased in 12 mg/kg group after fifteen days(P < 0.05) and was restrained in the high dose group(60 mg/kg) in thirty days(P < 0.05), but the contents of gamma-GT were arrested with different doses dependently in the testis(P < 0.05). Fenvalerate caused dose-dependent reduction in sperm head counts and daily sperm production, which markedly reduced at the doses of > or = 12 mg/kg groups(P < 0.01). CONCLUSIONS: Fenvalerate has obvious reproductive toxicity on male rats and can change their serum and testis homogenate levels of sex hormone or activity of testicular marked enzymes, which may be correlated with the impairment of Sertoli cell and spermatogenic epithelium.

[Effects of exposure to aluminum on some metal elements contents in hippocampus of rat].

OBJECTIVE: To investigate the effects of exposure to aluminum (Al) on Zn, Fe, Cu and Ca contents in hippocampus of rats. METHODS: AlCl(3).6H(2)O was administered orally through diet (8% of body weight) to SD rats at doses of 0 (A), 11.2 (B), 55.9 (C), 111.9 (D) mg Al(3+)/kg BW for successive 90 days. Then Al, Zn, Fe, Cu and Ca contents in hippocampi of rats were determined by atomic absorption spectrophotometry. RESULTS: After oral exposure to Al, the Al content in hippocampus of rat increased significantly with a remarkable dose-effect relationship (r = 0.731, P < 0.001), The Zn, Fe, Ca and Cu contents in these four groups are as follows: Group A (18.29 +/- 2.48, 24.86 +/- 1.97, 48.69 +/- 22.08, 4.53 +/- 0.99) mg/g, Group B (17.22 +/- 2.06, 27.54 +/- 2.87, 42.79 +/- 14.42, 4.06 +/- 0.41) mg/g, Group C (14.46 +/- 1.90, 20.18 +/- 2.79, 29.95 +/- 7.33, 3.98 +/- 0.25) mg/g, Group D (15.85 +/- 2.54, 20.96 +/- 2.83, 36.14 +/- 12.66, 4.53 +/- 0.58) mg/g. Compared with the control group A, the Zn and Fe contents in group B and group C decreased significantly (P < 0.05), and the Ca content in group C also decreased significantly. CONCLUSION: Oral exposure to Al may result in accumulation of Al in hippocampus of brain and thus affect some essential elements (Zn, Fe, Cu and Ca) contents in the hippocampus at different degrees.