Lateral Meniscal Root Displacement into the Popliteal Hiatus in a Multiligamentous Knee Injury: A Case Report.

CASE: A 45-year-old female patient presented with left knee pain after a ski accident. Magnetic Resonance Imaging (MRI) showed complete rupture of the anterior cruciate ligament, posterior cruciate ligament, proximal Medial Collateral Ligament (MCL), and medial patellofemoral ligament. The lateral meniscal root was torn with the posterior horn incarcerated superiorly into the popliteal hiatus, rendering high risk of plastic deformation. An unconventional 2-staged surgical approach was performed. CONCLUSION: Where risk of meniscal plastic deformation is high in the setting of multiligamentous knee injury (MLKI), diagnosis and careful surgical planning are central to a successful outcome. Treatment of intra-articular structures in operative stage 1 in MLKI is possible and was necessary in this case.

COVID-19 Diagnosis and SARS-CoV-2 Strain Identification by a Rapid, Multiplexed, Point-of-Care Antibody Microarray.

Antigen tests to detect SARS-CoV-2 have emerged as a promising rapid diagnostic method for COVID-19, but they are unable to differentiate between variants of concern (VOCs). Here, we report a rapid point-of-care test (POC-T), termed CoVariant-SPOT, that uses a set of antibodies that are either tolerant or intolerant to spike protein mutations to identify the likely SARS-CoV-2 strain concurrent with COVID-19 diagnosis using antibodies targeting the nucleocapsid protein. All reagents are incorporated into a portable, multiplexed, and sensitive diagnostic platform built upon a nonfouling polymer brush. To validate CoVariant-SPOT, we tested recombinant SARS-CoV-2 proteins, inactivated viruses, and nasopharyngeal swab samples from COVID-19 positive and negative individuals and showed that CoVariant-SPOT can readily distinguish between two VOCs: Delta and Omicron. We believe that CoVariant-SPOT can serve as a valuable adjunct to next-generation sequencing to rapidly identify variants using a scalable and deployable POC-T, thereby enhancing community surveillance efforts worldwide and informing treatment selection.

Phase I Study of Niraparib in Combination with Radium-223 for the Treatment of Metastatic Castrate-Resistant Prostate Cancer.

PURPOSE: To identify the safety of niraparib, a PARP inhibitor, in combination with Radium-223 for the treatment of metastatic castrate-resistant prostate cancer (mCRPC) in men without known BRCA mutations. PATIENTS AND METHODS: Men with progressive mCPRC following ≥1 line of androgen receptor (AR)-targeted therapy and bone metastases but no documented BRCA-1 or BRCA-2 alterations or bulky visceral disease were included. Niraparib dose was escalated in combination with standard dosing of Radium-223 using a time-to-event continual reassessment method. The highest dose level with a DLT probability <20% was defined as MTD. Secondary endpoints included PSA change and progression-free survival. Exploratory analyses included assessing DNA mutations found in ctDNA as well as gene expression changes assessed in whole blood samples. RESULTS: Thirty patients were treated with niraparib and radium-223: 13 patients received 100 mg, 12 received 200 mg, and 5 patients received 300 mg of niraparib. There were six DLT events: two (13%) for neutropenia, two (13%) for thrombocytopenia, whereas fatigue and nausea each occurred once (3%). Anemia (2/13%) and neutropenia (2/13%) were the most common grade 3 adverse events. For patients with prior chemotherapy exposure, the MTD was 100 mg, whereas the MTD for chemotherapy naïve patients was 200 mg. Whole blood gene expression of PAX5 and CD19 was higher in responders and ARG-1, IL2R, and FLT3 expression was higher in nonresponders. CONCLUSIONS: Combining niraparib with Radium-223 in patients with mCRPC was safe; however, further studies incorporating biomarkers will better elucidate the role of combinations of PARP inhibitors with DNA damaging and other agents.

Prognostic Value of GIMAP4 and Its Role in Promoting Immune Cell Infiltration into Tumor Microenvironment of Lung Adenocarcinoma.

GIMAPs are recognized as an important regulator in the carcinogenesis and development of lung cancer, but the function of GIMAP4 in the tumor microenvironment (TME) of lung cancers is unclear. In this study, we investigated the expression and variation of GIMAP4 in lung adenocarcinoma (LUAD), to explore its association with infiltration of immune cells. The Cancer Genome Atlas (TCGA) data and Gene Expression Omnibus (GEO) data were analyzed. Infiltration of immune cells was identified with TIMER (Tumor Immune Estimation Resource) and TISIDB (an integrated repository portal for tumor-immune system interactions). GIMAP4 expression declined in non-small-cell lung cancer (NSCLC), correlated with a poor overall survival (OS) in LUAD, indicating that GIMAP4 was a promising prognostic biomarker in LUAD. GIMAP4 mutation frequency was 1.76% in TCGA cohort and was relevant to the expression of immune components. TIMER and CIBERSORT analysis further confirmed that high GIMAP4 expression possibly promoted immune cell infiltration into the TME, with low GIMAP4 impairing the efficacy of immunotherapies targeting common immune check point inhibitors (ICI). GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analyses were performed to provide insights into biological processes involved in LUAD. GIMAP4 was expected to be a prognostic biomarker in LUAD and provides potential adjuvant or neoadjuvant therapeutic strategies for targeting ICIs.

Molecular lung cancer: How targeted therapies and personalized medicine are re-defining cancer care.

Lung cancer remains the leading cause of cancer death in the United States and is unfortunately still frequently diagnosed in the metastatic setting, where the disease is considered incurable. Nearly 30% of these cancers may be driven by specific mutations that promote tumor growth and proliferation. These mutations are observed more frequently in young patients without significant smoking history and in certain racial and ethnic backgrounds. The past 15 years have marked a revolution for patients with molecularly driven lung cancer as novel, oral, targeted therapies have been developed that demonstrate superior activity with substantially better toxicity profiles in comparison to chemotherapy. Consideration of molecular testing for a driver mutation is imperative for all providers caring for patients with a new suspected lung cancer diagnosis, as discovery of an actionable mutation will have dramatic implications in regards to patient survival and quality of life.

Multiplexed, quantitative serological profiling of COVID-19 from blood by a point-of-care test.

Highly sensitive, specific, and point-of-care (POC) serological assays are an essential tool to manage coronavirus disease 2019 (COVID-19). Here, we report on a microfluidic POC test that can profile the antibody response against multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigens-spike S1 (S1), nucleocapsid (N), and the receptor binding domain (RBD)-simultaneously from 60 µl of blood, plasma, or serum. We assessed the levels of antibodies in plasma samples from 31 individuals (with longitudinal sampling) with severe COVID-19, 41 healthy individuals, and 18 individuals with seasonal coronavirus infections. This POC assay achieved high sensitivity and specificity, tracked seroconversion, and showed good concordance with a live virus microneutralization assay. We can also detect a prognostic biomarker of severity, IP-10 (interferon-γ-induced protein 10), on the same chip. Because our test requires minimal user intervention and is read by a handheld detector, it can be globally deployed to combat COVID-19.

Isolation of extracellular vesicles with multivalent aptamers.

Extracellular vesicles (EVs) are lipid-enclosed submicron-sized vesicles that are secreted by all eukaryotic cells. EVs can selectively encapsulate tissue-specific small molecules from parent cells and efficiently deliver them to recipient cells. As signal mediators of intercellular communication, the molecules packaged in EVs play critical roles in the pathophysiology of diseases. In relevant clinical translation, EV contents have been used for cancer diagnosis and treatment monitoring. To further promote EV-based cancer liquid biopsy toward large-scale clinical implementation, the efficient and specific isolation of pure tumor-derived EVs from body fluids is a prerequisite. However, the existing EV isolation methods are unable to address certain technical challenges, such as lengthy procedures, low throughput, low specificity, heavy protein contamination, etc., and thus, new approaches for EV isolation are required. Here, we report a multivalent, long single-stranded aptamer with repeated units for EV enrichment and retrieval. After short incubation of biotin-labeled multivalent aptamers (MAs) with the samples, EVs can be quickly secured by MAs, anchored onto streptavidin-coated microspheres, and further retrieved via digestion of the DNA aptamer. Approximately 45% of EVs can be isolated from the spiked samples in 40 min with a depletion of 84.7% of albumin contamination. In addition, 93.1% of the isolated EVs can be retrieved via DNase-mediated aptamer degradation in 10 min for downstream molecular analyses. Our findings suggest that MAs can efficiently and specifically isolate EVs derived from malignant lymphocytes, and this simple method could facilitate the EV-centered study of acute lymphoblastic leukemia.

Conferring receptors on recipient cells with extracellular vesicles for targeted drug delivery.

Triple negative breast cancer (TNBC) is a heterogeneous subset of breast cancer characterized by its lack of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (HER2), which altogether prevents TNBC from being treated effectively. For many years, the treatment paradigms and overall survival of patients with TNBC have remained largely stagnant. Recent attempts to convert cold tumors to hot tumors by promoting antigen presentation have shown increased T cell infiltration and significantly induced immune responses for tumor killing. Inspired by this concept, the expression of specific targetable antigens on TNBC cells may further benefit relevant targeted drug delivery. In this study, we successfully conferred sufficient HER2 on the surface of TNBC MDA-MB-231 cells via simple EV-plasma membrane fusion with HER2+ extracellular vesicles (EV) derived from HER2 overexpressing BT-474 cells. Subsequently, anti-HER2 antibody conjugated paclitaxel-loaded liposomes were used for HER2-targeted drug delivery. Our findings demonstrated this HER2 grafting, in conjunction with targeted drug delivery, can improve the treatment efficacy in vitro and in vivo. This novel approach represents a facile method of altering cell membrane antigen presentation via convenient EVs uptake and may pave the way for the burgeoning wave of targeted therapy and/or immunotherapy.

Factors influencing the measurement of the secretion rate of extracellular vesicles.

Extracellular vesicles (EVs) are cell-derived vesicles which encapsulate a variety of molecules. Numerous studies have demonstrated EVs as signaling mediators of intercellular communication and are heavily involved under physiological and pathological conditions. In translational medicine, EVs have been used for disease diagnosis and treatment monitoring. EVs as natural nanocarriers for drug delivery and therapeutic EVs are also under intense investigation. While still in its infancy, relevant EV studies have been growing. For EV-centered research to thrive, a few fundamental unanswered questions, such as EV biogenesis, EV secretion rate (SR), EV content sorting mechanisms, etc. require further investigation. In this study, we measured the SR of EVs derived from 6 cancerous cell lines. Several factors that may interfere with EV secretion, isolation, and storage were also investigated. Our results show that the SR of EVs derived from various cancer cells was significantly different, indicating a heterogeneous EV secretion behavior among cell types. Moreover, 5 different drugs that interfere with cellular metabolism significantly influenced EV release. In addition, we found that (1) more EVs can be harvested at 24 h compared to 48 h of serum-free cell culture with a similar degree of FBS contamination; (2) filtration of the cell culture supernatant with a 0.22 µm filter causes ∼70% loss of EVs; (3) the isolation efficiency of EVs with the prevalent ultracentrifugation is only ∼14%; (4) storage at 4 °C for 3 days causes ∼21% loss of EVs. Overall, our findings provide a guideline for proper EV collection and storage in laboratory settings.

Primary Dural Diffuse Large B-cell Lymphoma: A Comprehensive Review of Survival and Treatment Outcomes.

Primary dural diffuse large B-cell lymphoma (PD-DLBCL) is a rare and aggressive B-cell non-Hodgkin lymphoma that can present in intracranial or intraspinal locations. Although the optimal management is unknown, PD-DLBCL therapy is often mirrored after primary central nervous system lymphoma therapy and aggressive treatment with a high dose methotrexate-based regimen is frequently used. Our comprehensive, retrospective study of 24 reported cases of PD-DLBCL provide the most complete analysis of this rare disease including data on biology, treatment outcomes, and survival. Our findings demonstrate good outcomes following induction treatment with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone), suggesting that these cases can be treated as DLBCL rather than primary central nervous system lymphoma, obviating the need for more aggressive and toxic approaches. The durable responses following R-CHOP also confirm that PD-DLBCL is not protected by the blood brain barrier.

Isolation and Retrieval of Extracellular Vesicles for Liquid Biopsy of Malignant Ground-Glass Opacity.

Extracellular vesicles (EVs) are cell-released vesicles of submicrometer size. EVs contain a tissue-specific signature wherein a variety of proteins and nucleic acids are selectively packaged. Recent studies validate that EVs can be used for cancer diagnostics, staging, and treatment monitoring. EV-related clinical translation requires effective EV isolation as a prerequisite. However, lengthy procedures, low yield, low throughput, and high levels of contaminants disqualify the existing isolation approaches for large-scale clinical use. Hence, new approaches for rapid, efficient, and low-cost isolation of EVs in high purity for flexible analyses of the diverse contents in real-world clinical settings are highly desired yet are currently unavailable. Here, we report the effective use of heparin/polymer-coated microspheres (HPM) for EV isolation and retrieval. Approximately 81% of EVs can be isolated from plasma in 1 h with depletion of ∼99.5% plasma protein and nucleic acid contaminants, and 72% of isolated EVs can be retrieved with saline in 5 min for various cargo analyses. This approach was further validated with clinical samples derived from patients with malignant ground-glass opacity (GGO). In eight patients, the mutation concordance between EV DNA and tissue DNA is 39.8%. The prevalence and mutation count of EGFR, TP53, and NF1 are higher than those of other oncogenes and antioncogenes that are intensely associated with lung adenocarcinoma. Moreover, different mutation prevalence and patterns between smokers and nonsmokers can be observed. Our findings suggest that the combination of HPM assay and targeted sequencing of EV DNA could be translated in the differential diagnosis of malignant GGO with short turnaround time.

Dysregulated mTORC1 renders cells critically dependent on desaturated lipids for survival under tumor-like stress.

Solid tumors exhibit heterogeneous microenvironments, often characterized by limiting concentrations of oxygen (O2), glucose, and other nutrients. How oncogenic mutations alter stress response pathways, metabolism, and cell survival in the face of these challenges is incompletely understood. Here we report that constitutive mammalian target of rapamycin complex 1 (mTORC1) activity renders hypoxic cells dependent on exogenous desaturated lipids, as levels of de novo synthesized unsaturated fatty acids are reduced under low O2. Specifically, we demonstrate that hypoxic Tsc2(-/-) (tuberous sclerosis complex 2(-/-)) cells deprived of serum lipids exhibit a magnified unfolded protein response (UPR) but fail to appropriately expand their endoplasmic reticulum (ER), leading to inositol-requiring protein-1 (IRE1)-dependent cell death that can be reversed by the addition of unsaturated lipids. UPR activation and apoptosis were also detected in Tsc2-deficient kidney tumors. Importantly, we observed this phenotype in multiple human cancer cell lines and suggest that cells committed to unregulated growth within ischemic tumor microenvironments are unable to balance lipid and protein synthesis due to a critical limitation in desaturated lipids.

Endothelial HIF-2α regulates murine pathological angiogenesis and revascularization processes.

Localized tissue hypoxia is a consequence of vascular compromise or rapid cellular proliferation and is a potent inducer of compensatory angiogenesis. The oxygen-responsive transcriptional regulator hypoxia-inducible factor 2α (HIF-2α) is highly expressed in vascular ECs and, along with HIF-1α, activates expression of target genes whose products modulate vascular functions and angiogenesis. However, the mechanisms by which HIF-2α regulates EC function and tissue perfusion under physiological and pathological conditions are poorly understood. Using mice in which Hif2a was specifically deleted in ECs, we demonstrate here that HIF-2α expression is required for angiogenic responses during hindlimb ischemia and for the growth of autochthonous skin tumors. EC-specific Hif2a deletion resulted in increased vessel formation in both models; however, these vessels failed to undergo proper arteriogenesis, resulting in poor perfusion. Analysis of cultured HIF-2α-deficient ECs revealed cell-autonomous increases in migration, invasion, and morphogenetic activity, which correlated with HIF-2α-dependent expression of specific angiogenic factors, including delta-like ligand 4 (Dll4), a Notch ligand, and angiopoietin 2. By stimulating Dll4 signaling in cultured ECs or restoring Dll4 expression in ischemic muscle tissue, we rescued most of the HIF-2α-dependent EC phenotypes in vitro and in vivo, emphasizing the critical role of Dll4/Notch signaling as a downstream target of HIF-2α in ECs. These results indicate that HIF-1α and HIF-2α fulfill complementary, but largely nonoverlapping, essential functions in pathophysiological angiogenesis.