Imaging the Oocysts in the Midgut of the Mosquito

● Learn how to dissect the midgut from the mosquito ○ Protocol on how to conduct the dissection ● Produce a portfolio of high end microscopy images of the oocysts inside of the midgut ○ Using Leica SP5 Point Scanning Confocal (Analytical Imaging Facility at Einstein College of Medicine) ○ Reconstruct the oocyst in 3D ○ To further be used in Dr. Kami Kim’s research ● Develop an image analysis protocol to automatically count each individual oocyst ○ Using Volocity analysis software (PerkinElmer) ○ Gather total counts and area measurements of individual oocysts ○ Written for future use ○ To further be used in Dr. Kami Kim’s researc

Imaging the Oocysts in the Midgut of the Mosquito

● Learn how to dissect the midgut from the mosquito ○ Protocol on how to conduct the dissection ● Produce a portfolio of high end microscopy images of the oocysts inside of the midgut ○ Using Leica SP5 Point Scanning Confocal (Analytical Imaging Facility at Einstein College of Medicine) ○ Reconstruct the oocyst in 3D ○ To further be used in Dr. Kami Kim’s research ● Develop an image analysis protocol to automatically count each individual oocyst ○ Using Volocity analysis software (PerkinElmer) ○ Gather total counts and area measurements of individual oocysts ○ Written for future use ○ To further be used in Dr. Kami Kim’s researc

Modeling Microorganism Transmission with Madagascar Hissing Cockroaches: An Inquiry Activity

Students will test Madagascar hissing cockroach’s capacity as a vector for transmission of microorganisms. By comparing a cockroach exposed to human contact (handled by students) and a cockroach with limited exposure (not handled), students can assess the ability of cockroaches to transmit microorganisms from one location (hands) to another (agar plate where the microorganism will be grown). This will allow students to determine if the Madagascar hissing cockroach, the classroom pet, is a potential vector for microorganisms. Students then will be able to question and relate the concept of insects and objects as vectors for common pathogen transfer

Targeted GAS6 Delivery to the CNS Protects Axons from Damage during Experimental Autoimmune Encephalomyelitis

Growth arrest-specific protein 6 (GAS6) is a soluble agonist of the TYRO3, AXL, MERTK (TAM) family of receptor tyrosine kinases identified to have anti-inflammatory, neuroprotective, and promyelinating properties. During experimental autoimmune encephalomyelitis (EAE), wild-type (WT) mice demonstrate a significant induction of Gas6, Axl, and Mertk but not Pros1 or Tyro3 mRNA. We tested the hypothesis that intracerebroventricular delivery of GAS6 directly into the CNS of WT mice during myelin oligodendrocyte glycoprotein (MOG)-induced EAE would improve the clinical course of disease relative to artificial CSF (ACSF)-treated mice. GAS6 did not delay disease onset, but significantly reduced the clinical scores during peak and chronic EAE. Mice receiving GAS6 for 28 d had preserved SMI31(+) neurofilament immunoreactivity, significantly fewer SMI32(+) axonal swellings and spheroids and less demyelination relative to ACSF-treated mice. Alternate-day subcutaneous IFNß injection did not enhance GAS6 treatment effectiveness. Gas6(-/-) mice sensitized with MOG35-55 peptide exhibit higher clinical scores during late peak to early chronic disease, with significantly increased SMI32(+) axonal swellings and Iba1(+) microglia/macrophages, enhanced expression of several proinflammatory mRNA molecules, and decreased expression of early oligodendrocyte maturation markers relative to WT mouse spinal cords with scores for 8 consecutive days. During acute EAE, flow cytometry showed significantly more macrophages but not T-cell infiltrates in Gas6(-/-) spinal cords than WT spinal cords. Our data are consistent with GAS6 being protective during EAE by dampening the inflammatory response, thereby preserving axonal integrity and myelination.This work was supported by the National Multiple Sclerosis Society (Grant RG 4046-A6) and the National Institutes of Health (Grant R21 NS079144-01, Neuropathology Training Grant T32 NS007098, and Cellular and Molecular Biology and Genetics Training Grant T32 GM007491).Peer Reviewe

HIV Nef and Antiretroviral Therapy Have an Inhibitory Effect on Autophagy in Human Astrocytes that May Contribute to HIV-Associated Neurocognitive Disorders

A significant number of people living with HIV (PLWH) develop HIV-associated neurocognitive disorders (HAND) despite highly effective antiretroviral therapy (ART). Dysregulated macroautophagy (autophagy) is implicated in HAND pathogenesis. The viral protein Nef, expressed even with suppressive ART, and certain antiretrovirals affect autophagy in non-CNS cells. Astrocytes, vital for CNS microenvironment homeostasis and neuronal health, require autophagy for their own homeostasis. We hypothesized that extracellular Nef and/or ART impact astrocyte autophagy, thus contributing to HAND. We studied in-bulk and selective autophagic flux in primary human astrocytes treated with extracellular Nef and/or a combination of tenofovir+emtricitabine+raltegravir (ART) using Western blotting, a tandem fluorescent LC3 reporter, and transmission electron microscopy/morphometry. We show that after 24 h treatment, Nef and ART decrease autophagosomes through different mechanisms. While Nef accelerates autophagosome degradation without inducing autophagosome formation, ART inhibits autophagosome formation. Combination Nef+ART further depletes autophagosomes by inducing both abnormalities. Additionally, extracellular Nef and/or ART inhibit lysosomal degradation of p62, indicating Nef and/or ART affect in-bulk and selective autophagy differently. Dysregulation of both autophagic processes is maintained after 7 days of Nef and/or ART treatment. Persistent autophagy dysregulation due to chronic Nef and/or ART exposure may ultimately result in astrocyte and neuronal dysfunction, contributing to HAND

Gas6 Enhances Axonal Ensheathment by MBP Membranous Processes in Human DRG/OL Promyelinating Co-Cultures

The molecular requirements for human myelination are incompletely defined, and further study is needed to fully understand the cellular mechanisms involved during development and in demyelinating diseases. We have established a human co-culture model to study myelination. Our earlier observations showed that addition of human γ-carboxylated growth-arrest-specific protein 6 (Gas6) to human oligodendrocyte progenitor cell (OPC) cultures enhanced their survival and maturation. Therefore, we explored the effect of Gas6 in co-cultures of enriched OPCs plated on axons of human fetal dorsal root ganglia explant. Gas6 significantly enhanced the number of myelin basic protein-positive (MBP + ) oligodendrocytes with membranous processes parallel with and ensheathing axons relative to co-cultures maintained in defined medium only for 14 days. Gas6 did not increase the overall number of MBP + oligodendrocytes/culture; however, it significantly increased the length of MBP + oligodendrocyte processes in contact with and wrapping axons. Multiple oligodendrocytes were in contact with a single axon, and several processes from one oligodendrocyte made contact with one or multiple axons. Electron microscopy supported confocal Z-series microscopy demonstrating axonal ensheathment by MBP + oligodendrocyte membranous processes in Gas6-treated co-cultures. Contacts between the axonal and oligodendrocyte membranes were evident and multiple wraps of oligodendrocyte membrane around the axon were visible supporting a model system in which to study events in human myelination and aspects of non-compact myelin formation

Development and Characterization of a Genetic Mouse Model of KRAS Mutated Colorectal Cancer

Patients with KRAS mutated colorectal cancer (CRC) represent a cohort with unmet medical needs, with limited options of FDA-approved therapies. Representing 40–45% of all CRC patients, they are considered ineligible to receive anti-EGFR monoclonal antibodies that have added a significant therapeutic benefit for KRAS wild type CRC patients. Although several mouse models of CRC have been developed during the past decade, one genetically resembling the KRAS mutated CRC is yet to be established. In this study C57 BL/6 mice with truncated adenomatous polyposis coli (APC) floxed allele was crossed with heterozygous KRAS floxed outbred mice to generate an APCf/f KRAS+/f mouse colony. In another set of breeding, APC floxed mice were crossed with CDX2-Cre-ERT2 mice and selected for APCf/f CDX2-Cre-ERT2 after the second round of inbreeding. The final model of the disease was generated by the cross of the two parental colonies and viable APC f/f KRAS +/f CDX2-Cre-ERT2 (KPC: APC) were genotyped and characterized. The model animals were tamoxifen (TAM) induced to generate tumors. Micro-positron emission tomography (PET) scan was used to detect and measure tumor volume and standard uptake value (SUV). Hematoxylin and eosin (H&E) staining was performed to establish neoplasm and immunohistochemistry (IHC) was performed to determine histological similarities with human FFPE biopsies. The MSI/microsatellite stable (MSS) status was determined. Finally, the tumors were extensively characterized at the molecular level to establish similarities with human CRC tumors. The model KPC: APC animals are conditional mutants that developed colonic tumors upon induction with tamoxifen in a dose-dependent manner. The tumors were confirmed to be malignant within four weeks of induction by H&E staining and higher radioactive [18F] fluoro-2-deoxyglucose (FDG) uptake (SUV) in micro-PET scan. Furthermore, the tumors histologically and molecularly resembled human colorectal carcinoma. Post tumor generation, the KPC: APC animals died of cachexia and rectal bleeding. Implications: This model is an excellent preclinical platform to molecularly characterize the KRAS mutated colorectal tumors and discern appropriate therapeutic strategies to improve disease management and overall survival

The effect of bone morphogenetic protein-2 on osteosarcoma metastasis.

PURPOSE:Bone Morphogenetic Protein-2 (BMP-2) may offer the potential to enhance allograft-host osseous union in limb-salvage surgery following osteosarcoma resection. However, there is concern regarding the effect of locally applied BMP-2 on tumor recurrence and metastasis. The purpose of this project was to evaluate the effect of exogenous BMP-2 on osteosarcoma migration and invasion across a panel of tumor cell lines in vitro and to characterize the effect of BMP-2 on pulmonary osteosarcoma metastasis within a xenograft model. EXPERIMENTAL DESIGN:The effect of BMP-2 on in vitro tumor growth and development was assessed across multiple standard and patient-derived xenograft osteosarcoma cell lines. Tumor migration capacity, invasion, and cell proliferation were characterized. In addition, the effect on metastasis was measured using a xenograft model following tail-vein injection. The effect of exogenous BMP-2 on the development of metastases was measured following both single and multiple BMP-2 administrations. RESULTS:There was no significant difference in migration capacity, invasion, or cell proliferation between the BMP-2 treated and the untreated osteosarcoma cell lines. There was no significant difference in pulmonary metastases between either the single-dose or multi-dose BMP-2 treated animals and the untreated control animals. CONCLUSIONS:In the model systems tested, the addition of BMP-2 does not increase osteosarcoma proliferation, migration, invasion, or metastasis to the lungs

Effects of BMP-2 on the proliferation rate of different osteosarcoma cell lines.

<p>Four different amounts of BMP-2 were added and the proliferation rate was measured at 24, 48, 72, and 96 hours. (A) 143B (B) U2OS (C) HOS (D) MG63 (E) SaOS-2 (F) LM7 (G) OS17 (H) OS31. There were no changes in proliferation rates with increasing amounts of BMP-2.</p

Single-dose BMP-2 treated animals bearing tumor (SaOS-LM7) compared to control animals.

<p>There was no significant difference in body weight, lung weight, or lung weight/body weight ratio between the two groups.</p