Authors: Amano M, Bulut H, Tamiya S, Nakamura T, Koh Y, Mitsuya H
Journal: Sci Rep. 2019 Jul 8;9(1):9806. doi: 10.1038/s41598-019-46082-2.
Accumulation of amino acid (AA) insertions/substitutions are observed in the Gag-protein of HIV-1 variants resistant to HIV-1 protease inhibitors. Here, we found that HIV-1 carrying AA insertions in capsid protein (CA) undergoes aberrant CA degradation. When we generated recombinant HIV-1s (rHIV-1s) containing 19-AAs in Gag, such insertions caused significant CA degradation, which initiated in CA's C-terminal. Such rHIV-1s had remarkable morphological abnormality, decreased infectivity, and no replicative ability, which correlated with levels of CA degradation. The CA degradation observed was energy-independent and had no association with cellular/viral proteolytic mechanisms, suggesting that the CA degradation occurs due to conformational/structural incompatibility caused by the 19-AA insertions. The incorporation of degradation-prone CA into the wild-type CA resulted in significant disruption of replication competence in "chimeric" virions. The data should allow better understanding of the dynamics and mechanisms of CA decomposition/degradation and retroviral uncoating, which may lead to new approach for antiretroviral modalities.
Authors: Nagi SS, Marshall AG, Makdani A, Jarocka E, Liljencrantz J, Ridderström M, Shaikh S, O'Neill F, Saade D, Donkervoort S, Foley AR, Minde J, Trulsson M, Cole J, Bönnemann CG, Chesler AT, Bushnell MC, McGlone F, Olausson H
Journal: Sci Adv. 2019 Jul 3;5(7):eaaw1297. doi: 10.1126/sciadv.aaw1297. eCollection 2019 Jul.
The canonical view is that touch is signaled by fast-conducting, thickly myelinated afferents, whereas pain is signaled by slow-conducting, thinly myelinated ("fast" pain) or unmyelinated ("slow" pain) afferents. While other mammals have thickly myelinated afferents signaling pain (ultrafast nociceptors), these have not been demonstrated in humans. Here, we performed single-unit axonal recordings (microneurography) from cutaneous mechanoreceptive afferents in healthy participants. We identified A-fiber high-threshold mechanoreceptors (A-HTMRs) that were insensitive to gentle touch, encoded noxious skin indentations, and displayed conduction velocities similar to A-fiber low-threshold mechanoreceptors. Intraneural electrical stimulation of single ultrafast A-HTMRs evoked painful percepts. Testing in patients with selective deafferentation revealed impaired pain judgments to graded mechanical stimuli only when thickly myelinated fibers were absent. This function was preserved in patients with a loss-of-function mutation in mechanotransduction channel PIEZO2. These findings demonstrate that human mechanical pain does not require PIEZO2 and can be signaled by fast-conducting, thickly myelinated afferents.
Authors: Fujita K, Chakarov S, Kobayashi T, Sakamoto K, Voisin B, Duan K, Nakagawa T, Horiuchi K, Amagai M, Ginhoux F, Nagao K
Journal: Proc Natl Acad Sci U S A. 2019 Jul 1. pii: 201818907. doi: 10.1073/pnas.1818907116. [Epub ahead of print]
Conventional dendritic cells (cDCs) derive from bone marrow (BM) precursors that undergo cascades of developmental programs to terminally differentiate in peripheral tissues. Pre-cDC1s and pre-cDC2s commit in the BM to each differentiate into CD8α+/CD103+ cDC1s and CD11b+ cDC2s, respectively. Although both cDCs rely on the cytokine FLT3L during development, mechanisms that ensure cDC accessibility to FLT3L have yet to be elucidated. Here, we generated mice that lacked a disintegrin and metalloproteinase (ADAM) 10 in DCs (Itgax-cre × Adam10-fl/fl; ADAM10∆DC) and found that ADAM10 deletion markedly impacted splenic cDC2 development. Pre-cDC2s accumulated in the spleen with transcriptomic alterations that reflected their inability to differentiate and exhibited abrupt failure to survive as terminally differentiated cDC2s. Induced ADAM10 ablation also led to the reduction of terminally differentiated cDC2s, and restoration of Notch signaling, a major pathway downstream of ADAM10, only modestly rescued them. ADAM10∆DC BM failed to generate cDC2s in BM chimeric mice with or without cotransferred ADAM10-sufficient BM, indicating that cDC2 development required cell-autonomous ADAM10. We determined cDC2s to be sources of soluble FLT3L, as supported by decreased serum FLT3L concentration and the retention of membrane-bound FLT3L on cDC2 surfaces in ADAM10∆DC mice, and by demonstrating the release of soluble FLT3L by cDC2 in ex vivo culture supernatants. Through in vitro studies utilizing murine embryonic fibroblasts, we determined FLT3L to be a substrate for ADAM10. These data collectively reveal cDC2s as FLT3L sources and highlight a cell-autonomous mechanism that may enhance FLT3L accessibility for cDC2 development and survival.
Authors: Ferris AL, Wells DW, Guo S, Del Prete GQ, Swanstrom AE, Coffin JM, Wu X, Lifson JD, Hughes SH
Journal: PLoS Pathog. 2019 Jul 10;15(7):e1007869. doi: 10.1371/journal.ppat.1007869. eCollection 2019 Jul.
Clonal expansion of HIV infected cells plays an important role in the formation and persistence of the reservoir that allows the virus to persist, in DNA form, despite effective antiretroviral therapy. We used integration site analysis to ask if there is a similar clonal expansion of SIV infected cells in macaques. We show that the distribution of HIV and SIV integration sites in vitro is similar and that both viruses preferentially integrate in many of the same genes. We obtained approximately 8000 integration sites from blood samples taken from SIV-infected macaques prior to the initiation of ART, and from blood, spleen, and lymph node samples taken at necropsy. Seven clones were identified in the pre-ART samples; one persisted for a year on ART. An additional 100 clones were found only in on-ART samples; a number of these clones were found in more than one tissue. The timing and extent of clonal expansion of SIV-infected cells in macaques and HIV-infected cells in humans is quite similar. This suggests that SIV-infected macaques represent a useful model of the clonal expansion of HIV infected cells in humans that can be used to evaluate strategies intended to control or eradicate the viral reservoir.
Authors: Staunton JR, So WY, Paul CD, Tanner K
Journal: Proc Natl Acad Sci U S A. 2019 Jul 2. pii: 201814271. doi: 10.1073/pnas.1814271116. [Epub ahead of print]
Mechanical homeostasis describes how cells sense physical cues from the microenvironment and concomitantly remodel both the cytoskeleton and the surrounding extracellular matrix (ECM). Such feedback is thought to be essential to healthy development and maintenance of tissue. However, the nature of the dynamic coupling between microscale cell and ECM mechanics remains poorly understood. Here we investigate how and whether cells remodel their cortex and basement membrane to adapt to their microenvironment. We measured both intracellular and extracellular viscoelasticity, generating a full factorial dataset on five cell lines in two ECMs subjected to four cytoskeletal drug treatments at two time points. Nonmalignant breast epithelial cells show a similar viscoelasticity to that measured for the local ECM when cultured in 3D laminin-rich ECM. In contrast, the malignant counterpart is stiffer than the local environment. We confirmed that other mammary cancer cells embedded in tissue-mimetic hydrogels are nearly four-fold stiffer than the surrounding ECM. Perturbation of actomyosin did not yield uniform responses but instead depended on the cell type and chemistry of the hydrogel. The observed viscoelasticity of both ECM and cells were well described by power laws in a frequency range that governs single filament cytoskeletal dynamics. Remarkably, the intracellular and extracellular power law parameters for the entire dataset collectively fall onto two parallel master curves described by just two parameters. Our work shows that tumor cells are mechanically plastic to adapt to many environments and reveals dynamical scaling behavior in the microscale mechanical responses of both cells and ECM.
Authors: Solinski HJ, Dranchak P, Oliphant E, Gu X, Earnest TW, Braisted J, Inglese J, Hoon MA
Journal: Sci Transl Med. 2019 Jul 10;11(500). pii: eaav5464. doi: 10.1126/scitranslmed.aav5464.
There is a major clinical need for new therapies for the treatment of chronic itch. Many of the molecular components involved in itch neurotransmission are known, including the neuropeptide NPPB, a transmitter required for normal itch responses to multiple pruritogens in mice. Here, we investigated the potential for a novel strategy for the treatment of itch that involves the inhibition of the NPPB receptor NPR1 (natriuretic peptide receptor 1). Because there are no available effective human NPR1 (hNPR1) antagonists, we performed a high-throughput cell-based screen and identified 15 small-molecule hNPR1 inhibitors. Using in vitro assays, we demonstrated that these compounds specifically inhibit hNPR1 and murine NPR1 (mNPR1). In vivo, NPR1 antagonism attenuated behavioral responses to both acute itch- and chronic itch-challenged mice. Together, our results suggest that inhibiting NPR1 might be an effective strategy for treating acute and chronic itch.
Authors: Shrestha D, Rahman ML, Hinkle SN, Workalemahu T, Tekola-Ayele F
Journal: Obesity (Silver Spring). 2019 Jul;27(7):1150-1160. doi: 10.1002/oby.22499.
OBJECTIVE: Associations between maternal genetic risk for obesity and fetal weight were examined at the end of the first (13 weeks 6 days), second (27 weeks 6 days), and third (40 weeks 0 days) trimesters of pregnancy among four race/ethnic groups in the US.
METHODS: For 603 white, 591 black, 535 Hispanic, and 216 Asian women, maternal genetic risk score (GRS) was calculated as the sum of 189 BMI-increasing alleles and was categorized into high or low GRS. Associations between GRS (continuous and categorical) and estimated fetal weight were tested overall and stratified by prepregnancy BMI, gestational weight gain (GWG), and fetal sex.
RESULTS: High GRS compared with low GRS was associated with increased fetal weight at the end of the second (β: 22.7 g; 95% CI: 2.4-43.1; P = 0.03) and third trimesters (β: 88.3 g; 95% CI: 9.0-167.6; P = 0.03) among Hispanic women. The effect of GRS was stronger among Hispanic women with normal prepregnancy weight, adequate first trimester GWG, or inadequate second trimester GWG (P < 0.05). Among Asian women, high GRS was associated with increased weight among male fetuses but decreased weight among female fetuses (P < 0.05).
CONCLUSIONS: Maternal obesity genetic risk was associated with fetal weight with potential effect modifications by maternal prepregnancy BMI, GWG, and fetal sex.
Authors: Farfel-Becker T, Roney JC, Cheng XT, Li S,Cuddy SR, Sheng ZH
Journal: Cell Rep. 2019 Jul 2;28(1):51-64.e4. doi: 10.1016/j.celrep.2019.06.013.
Neurons face the challenge of maintaining cellular homeostasis through lysosomal degradation. While enzymatically active degradative lysosomes are enriched in the soma, their axonal trafficking and positioning and impact on axonal physiology remain elusive. Here, we characterized axon-targeted delivery of degradative lysosomes by applying fluorescent probes that selectively label active forms of lysosomal cathepsins D, B, L, and GCase. By time-lapse imaging of cortical neurons in microfluidic devices and standard dishes, we reveal that soma-derived degradative lysosomes rapidly influx into distal axons and target to autophagosomes and Parkinson disease-related α-synuclein cargos for local degradation. Impairing lysosome axonal delivery induces an aberrant accumulation of autophagosomes and α-synuclein cargos in distal axons. Our study demonstrates that the axon is an active compartment for local degradation and reveals fundamental aspects of axonal lysosomal delivery and maintenance. Our work establishes a foundation for investigations into axonal lysosome trafficking and functionality in neurodegenerative diseases.
Authors: Yao C, Sun HW, Lacey NE, Ji Y, Moseman EA, Shih HY, Heuston EF, Kirby M, Anderson S, Cheng J, Khan O, Handon R, Reilley J, Fioravanti J, Hu J, Gossa S, Wherry EJ, Gattinoni L, McGavern DB, O'Shea JJ, Schwartzberg PL, Wu T
Journal: Nat Immunol. 2019 Jul;20(7):890-901. doi: 10.1038/s41590-019-0403-4.
Progenitor-like CD8+ T cells mediate long-term immunity to chronic infection and cancer and respond potently to immune checkpoint blockade. These cells share transcriptional regulators with memory precursor cells, including T cell-specific transcription factor 1 (TCF1), but it is unclear whether they adopt distinct programs to adapt to the immunosuppressive environment. By comparing the single-cell transcriptomes and epigenetic profiles of CD8+ T cells responding to acute and chronic viral infections, we found that progenitor-like CD8+ T cells became distinct from memory precursor cells before the peak of the T cell response. We discovered a coexpression gene module containing Tox that exhibited higher transcriptional activity associated with more abundant active histone marks in progenitor-like cells than memory precursor cells. Moreover, thymocyte selection-associated high mobility group box protein TOX (TOX) promoted the persistence of antiviral CD8+ T cells and was required for the programming of progenitor-like CD8+ T cells. Thus, long-term CD8+ T cell immunity to chronic viral infection requires unique transcriptional and epigenetic programs associated with the transcription factor TOX.
Authors: Cui J, Zhang Q, Song Q, Wang H, Dmitriev P, Sun M, Cao X, Wang Y, Guo L, Indig I, Rosenblum J, Ji C, Cao D, Yang K, Gilbert MR, Yao Y, Zhuang Z
Journal: Neuro Oncol. 2019 Jul 4. pii: noz117. doi: 10.1093/neuonc/noz117. [Epub ahead of print]
BACKGROUND: Glioblastoma survival remains unchanged despite continuing therapeutic innovation. Herein, we aim to 1) develop CAR-T cells with a specificity to a unique antigen, carbonic anhydrase IX (CAIX), which is expressed in the hypoxic microenvironment characteristic of glioblastoma, and 2) demonstrate its efficacy with limited off-target effects.
METHODS: First we demonstrated expression of CAIX in patient-derived glioblastoma samples and available databases. CAR-T cells were generated against CAIX and efficacy was assessed in four glioblastoma cell lines and two glioblastoma stem cell lines. Cytotoxicity of anti-CAIX CAR-T cells was assessed via IFN-γ, TNF-α, and IL-2 levels when co-cultured with tumor cells. Finally, we assessed efficacy of direct intra-tumoral injection of the anti-CAIX CAR-T cells on an in vivo xenograft mouse model using the U251 fluorescent cell line. Tumor infiltrating lymphocyte analyses were performed.
RESULTS: We confirm that CAIX is highly expressed in glioblastoma from patients. We demonstrate that CAIX is a suitable target for CAR-T therapy using anti-CAIX CAR-T cells against glioblastoma in vitro and in vivo. In our mouse model, a 20% cure rate was observed without detectable systemic effects.
CONCLUSIONS: By establishing the specificity of CAIX under hypoxic conditions in glioblastoma and highlighting its efficacy as a target for CAR-T therapy, our data suggest that anti-CAIX CAR-T may be a promising strategy to treat glioblastoma. Direct intra-tumoral injection increases anti-CAIX CAR-T potency while limiting its off-target effects.