Decreased long-range temporal correlations in the resting-state functional magnetic resonance imaging blood-oxygen-level-dependent signal reflect motor sequence learning up to 2 weeks following training.

Decreased long-range temporal correlations (LRTC) in brain signals can be used to measure cognitive effort during task execution. Here, we examined how learning a motor sequence affects long-range temporal memory within resting-state functional magnetic resonance imaging signal. Using the Hurst exponent (HE), we estimated voxel-wise LRTC and assessed changes over 5 consecutive days of training, followed by a retention scan 12 days later. The experimental group learned a complex visuomotor sequence while a complementary control group performed tightly matched movements. An interaction analysis revealed that HE decreases were specific to the complex sequence and occurred in well-known motor sequence learning associated regions including left supplementary motor area, left premotor cortex, left M1, left pars opercularis, bilateral thalamus, and right striatum. Five regions exhibited moderate to strong negative correlations with overall behavioral performance improvements. Following learning, HE values returned to pretraining levels in some regions, whereas in others, they remained decreased even 2 weeks after training. Our study presents new evidence of HE's possible relevance for functional plasticity during the resting-state and suggests that a cortical subset of sequence-specific regions may continue to represent a functional signature of learning reflected in decreased long-range temporal dependence after a period of inactivity.

Intracellular Plasmodium aquaporin 2 is important for sporozoite production in the mosquito vector and malaria transmission.

Malaria remains a devastating disease and, with current measures failing to control its transmission, there is a need for novel interventions. A family of proteins that have long been pursued as potential intervention targets are aquaporins, which are channels facilitating the movement of water and other solutes across membranes. We identify an aquaporin in malaria parasites and demonstrate that it is important for completion of Plasmodium development in the mosquito vector. Disruption of AQP2 in the human parasite Plasmodium falciparum and the rodent parasite Plasmodium berghei blocks sporozoite production inside oocysts established on mosquito midguts, greatly limiting parasite infection of salivary glands and transmission to a new host. In vivo epitope tagging of AQP2 in P. berghei, combined with immunofluorescence assays, reveals that the protein is localized in vesicle-like organelles found in the cytoplasm of gametocytes, ookinetes, and sporozoites. The number of these organelles varies between individual parasites and lifecycle stages suggesting that they are likely part of a dynamic endomembrane system. Phylogenetic analysis confirms that AQP2 is unique to malaria and closely related parasites and most closely resembles intracellular aquaporins. Structure prediction analyses identify several unusual features, including a large accessory extracellular loop and an arginine-to-phenylalanine substitution in the selectivity filter principally determining pore function, a unique feature among known aquaporins. This in conjunction with the importance of AQP2 for malaria transmission suggests that AQP2 may be a fruitful target of antimalarial interventions.

Identification of genes required for Plasmodium gametocyte-to-sporozoite development in the mosquito vector.

Malaria remains one of the most devastating infectious diseases. Reverse genetic screens offer a powerful approach to identify genes and molecular processes governing malaria parasite biology. However, the complex regulation of gene expression and genotype-phenotype associations in the mosquito vector, along with sexual reproduction, have hindered the development of screens in this critical part of the parasite life cycle. To address this, we developed a genetic approach in the rodent parasite Plasmodium berghei that, in combination with barcode sequencing, circumvents the fertilization roadblock and enables screening for gametocyte-expressed genes required for parasite infection of the mosquito Anopheles coluzzii. Our results confirm previous findings, validating our approach for scaling up, and identify genes necessary for mosquito midgut infection, oocyst development, and salivary gland infection. These findings can aid efforts to study malaria transmission biology and to develop interventions for controlling disease transmission.

The globalizability of temporal discounting.

Economic inequality is associated with preferences for smaller, immediate gains over larger, delayed ones. Such temporal discounting may feed into rising global inequality, yet it is unclear whether it is a function of choice preferences or norms, or rather the absence of sufficient resources for immediate needs. It is also not clear whether these reflect true differences in choice patterns between income groups. We tested temporal discounting and five intertemporal choice anomalies using local currencies and value standards in 61 countries (N = 13,629). Across a diverse sample, we found consistent, robust rates of choice anomalies. Lower-income groups were not significantly different, but economic inequality and broader financial circumstances were clearly correlated with population choice patterns.

Spectrum of Atazanavir-Selected Protease Inhibitor-Resistance Mutations.

Ritonavir-boosted atazanavir is an option for second-line therapy in low- and middle-income countries (LMICs). We analyzed publicly available HIV-1 protease sequences from previously PI-naïve patients with virological failure (VF) following treatment with atazanavir. Overall, 1497 patient sequences were identified, including 740 reported in 27 published studies and 757 from datasets assembled for this analysis. A total of 63% of patients received boosted atazanavir. A total of 38% had non-subtype B viruses. A total of 264 (18%) sequences had a PI drug-resistance mutation (DRM) defined as having a Stanford HIV Drug Resistance Database mutation penalty score. Among sequences with a DRM, nine major DRMs had a prevalence >5%: I50L (34%), M46I (33%), V82A (22%), L90M (19%), I54V (16%), N88S (10%), M46L (8%), V32I (6%), and I84V (6%). Common accessory DRMs were L33F (21%), Q58E (16%), K20T (14%), G73S (12%), L10F (10%), F53L (10%), K43T (9%), and L24I (6%). A novel nonpolymorphic mutation, L89T occurred in 8.4% of non-subtype B, but in only 0.4% of subtype B sequences. The 264 sequences included 3 (1.1%) interpreted as causing high-level, 14 (5.3%) as causing intermediate, and 27 (10.2%) as causing low-level darunavir resistance. Atazanavir selects for nine major and eight accessory DRMs, and one novel nonpolymorphic mutation occurring primarily in non-B sequences. Atazanavir-selected mutations confer low-levels of darunavir cross resistance. Clinical studies, however, are required to determine the optimal boosted PI to use for second-line and potentially later line therapy in LMICs.

Progesterone rapidly alters the use of place and response memory during spatial navigation in female rats.

17ß-Estradiol (E2) and progesterone (P) influence place and response memory in female rats in spatial navigation tasks. Use of these memory systems is associated with the hippocampus and the dorsal striatum, respectively. Injections of E2 result in a well-established bias to use place memory, while much less is understood about the role of P. A total of 120 ovariectomized female rats were tested within a dual-solution T-maze task and treated with either low E2 (n = 24), high E2 (10 µg/kg; n = 24), or high E2 in combination with P (500 µg/kg) at three time points before testing: 15 min (n = 24), 1 h (n = 24), and 4 h (n = 24). Given alone, high E2 biases rats to the use of place memory, but this effect is reversed when P is given 1 h or 4 h before testing. This indicates that P may be playing an inhibitory role in the hippocampus during spatial tasks, which is consistent with past findings. Our findings show that P acts rapidly (within an hour) to affect performance during spatial tasks.

Integrase Strand Transfer Inhibitor Resistance in Integrase Strand Transfer Inhibitor-Naive Persons.

There has been no systematic review of the prevalence of transmitted integrase strand transfer inhibitor (INSTI) resistance. We systematically searched the English-language PubMed database and GenBank to identify studies published since 2010 reporting 50 or more INSTI-naive HIV-1-infected adults undergoing integrase genotyping. We extracted data related to country, sample year, specimen type, sequencing method, and subtype. For studies with sequences in GenBank, we determined the prevalence of three categories of INSTI-associated resistance mutations: (1) nonpolymorphic INSTI-selected drug resistance mutations (DRMs) that we refer to as surveillance DRMs; (2) rarely selected nonpolymorphic INSTI-associated DRMs; and (3) common polymorphic accessory INSTI-selected DRMs. A total of 103 studies met inclusion criteria including 75 studies in GenBank containing integrase sequences from 16,481 INSTI-naive persons. The median sample year was 2013 (interquartile range: 2008-2014). The prevalence of INSTI surveillance DRMs, rarely selected DRMs, and common polymorphic accessory INSTI-selected DRMs were 0.5%, 0.8%, and 6.2%, respectively. There was no association between the presence of nonpolymorphic surveillance DRM and region, sample year, or subtype. Two surveillance DRMs, E138K and R263K occurred in 0.15% and 0.10% of naive sequences, respectively. Several lines of evidence suggested that the 0.5% prevalence of surveillance DRMs partly reflects the cumulative natural occurrence of these mutations in the absence of selective drug pressure. There was an unexplained temporal increase in the proportion of sequences with polymorphic accessory mutations. The prevalence of INSTI-associated surveillance DRMs is low even in regions where INSTIs have been a major component of antiretroviral therapy for several years. The presence of INSTI-associated surveillance DRMs in INSTI-naive persons likely results from actual cases of transmitted INSTI resistance and from a low background level reflecting the cumulative rare natural occurrence of several nonpolymorphic mutations.

Inhibition of Phosphoinositide-3-Kinase Signaling Promotes the Stem Cell State of Trophoblast.

Trophoblast stem cells (TSCs) are a heterogeneous cell population despite the presence of fibroblast growth factor (FGF) and transforming growth factor ß (TGFB) as key growth factors in standard culture conditions. To understand what other signaling cascades control the stem cell state of mouse TSCs, we performed a kinase inhibitor screen and identified several novel pathways that cause TSC differentiation. Surprisingly, inhibition of phosphoinositide-3-kinase (PI3K) signaling increased the mRNA and protein expression of stem cell markers instead, and resulted in a tighter epithelial colony morphology and fewer differentiated cells. PI3K inhibition could not substitute for FGF or TGFB and did not affect phosphorylation of extracellular signal-regulated kinase, and thus acts independently of these pathways. Upon removal of PI3K inhibition, TSC transcription factor levels reverted to normal TSC levels, indicating that murine TSCs can reversibly switch between these two states. In summary, PI3K inhibition reduces the heterogeneity and seemingly heightens the stem cell state of TSCs as indicated by the simultaneous upregulation of multiple key marker genes and cell morphology. Stem Cells 2019;37:1307-1318.

United States Food and Drug Administration Regulation of Gene and Cell Therapies.

The United States (US) Food and Drug Administration (FDA) is a regulatory agency that has oversight for a wide range of products entering the US market, including gene and cell therapies. The regulatory approach for these products is similar to other medical products within the United States and consists of a multitiered framework of statutes, regulations, and guidance documents. Within this framework, there is considerable flexibility which is necessary due to the biological and technical complexity of these products in general. This chapter provides an overview of the US FDA regulatory oversight of gene and cell therapy products.

MSC-based product characterization for clinical trials: an FDA perspective.

Proposals submitted to the FDA for MSC-based products are undergoing a rapid expansion that is characterized by increased variability in donor and tissue sources, manufacturing processes, proposed functional mechanisms, and characterization methods. Here we discuss the diversity in MSC-based clinical trial product proposals and highlight potential challenges for clinical translation.

Murine spinotrapezius model to assess the impact of arteriolar ligation on microvascular function and remodeling.

The murine spinotrapezius is a thin, superficial skeletal support muscle that extends from T3 to L4, and is easily accessible via dorsal skin incision. Its unique anatomy makes the spinotrapezius useful for investigation of ischemic injury and subsequent microvascular remodeling. Here, we demonstrate an arteriolar ligation model in the murine spinotrapezius muscle that was developed by our research team and previously published(1-3). For certain vulnerable mouse strains, such as the Balb/c mouse, this ligation surgery reliably creates skeletal muscle ischemia and serves as a platform for investigating therapies that stimulate revascularization. Methods of assessment are also demonstrated, including the use of intravital and confocal microscopy. The spinotrapezius is well suited to such imaging studies due to its accessibility (superficial dorsal anatomy) and relative thinness (60-200 µm). The spinotrapezius muscle can be mounted en face, facilitating imaging of whole-muscle microvascular networks without histological sectioning. We describe the use of intravital microscopy to acquire metrics following a functional vasodilation procedure; specifically, the increase in arterilar diameter as a result of muscle contraction. We also demonstrate the procedures for harvesting and fixing the tissues, a necessary precursor to immunostaining studies and the use of confocal microscopy.

Prospective, multicenter, randomized, controlled study of anular repair in lumbar discectomy: two-year follow-up.

STUDY DESIGN: Prospective, multicenter, single-blind, randomized, controlled clinical study. OBJECTIVE: To investigate outcomes associated with repairing the anulus fibrosus after lumbar discectomy for the surgical management of herniated nucleus pulposus. SUMMARY OF BACKGROUND DATA: In patients undergoing discectomy, the incidence of reherniation ranges from 10% to 15%. Repair of the anulus fibrosus defect after lumbar discectomy may decrease the incidence of recurrent herniation for these patients. METHODS: A total of 750 patients were treated for herniated lumbar discs and randomly assigned in a 2:1 ratio to discectomy with the Xclose Tissue Repair System (Anulex Technologies, Minnetonka, MN) for anular repair (n = 500) or discectomy without anular repair (n = 250). Patient self-reported measures included visual analogue scales for leg and back pain, Oswestry Disability Index, and Short Form-12 Health Survey. Adverse events and subsequent reherniation surgical procedures were documented. Preoperative outcome measures were compared with follow-up visits at 2 weeks, 6 months, 1 year, and 2 years. RESULTS: Patient symptoms were improved after surgery in an equivalent manner in both study groups. In the overall study analysis, the rate of reherniation surgery was lower for Xclose patients at all follow-up time points, but these differences were not statistically significant. In patients with predominant leg pain, there was a significant reduction in reherniation risk at 3 and 6 months postoperation for patients receiving Xclose. A positive reduction was maintained through 2 years, with a clinically relevant risk reduction of 45%, although not statistically significant. Safety was demonstrated with similar improvements in patient outcomes and no difference in reported adverse events. CONCLUSION: Without a safe and effective method for closing the anulus fibrosus after discectomy, current practice has been to leave the anulus in a compromised state. This multicenter randomized study demonstrated that, while not statistically significant, anular repair reduced the need for subsequent reherniation surgery while retaining the benefits of discectomy with no increased risk for patients. LEVEL OF EVIDENCE: 1.

Balancing tissue and tumor formation in regenerative medicine.

A set of general principles can guide preclinical testing strategies for evaluating the tumorigenicity of regenerative medicine products.

Metastatic progression of prostate cancer and e-cadherin regulation by zeb1 and SRC family kinases.

Expression of E-cadherin is used to monitor the epithelial phenotype, and its loss is suggestive of epithelial-mesenchymal transition (EMT). EMT triggers tumor metastasis. Exit from EMT is marked by increased E-cadherin expression and is considered necessary for tumor growth at sites of metastasis; however, the mechanisms associated with exit from EMT are poorly understood. Herein are analyzed 185 prostate cancer metastases, with significantly higher E-cadherin expression in bone than in lymph node and soft tissue metastases. To determine the molecular mechanisms of regulation of E-cadherin expression, three stable isogenic cell lines from DU145 were derived that differ in structure, migration, and colony formation on soft agar and Matrigel. When injected into mouse tibia, the epithelial subline grows most aggressively, whereas the mesenchymal subline does not grow. In cultured cells, ZEB1 and Src family kinases decrease E-cadherin expression. In contrast, in tibial xenografts, E-cadherin RNA levels increase eight- to 10-fold despite persistent ZEB1 expression, and in all ZEB1-positive metastases (10 of 120), ZEB1 and E-cadherin proteins were co-expressed. These data suggest that transcriptional regulation of E-cadherin differs in cultured cells versus xenografts, which more faithfully reflect E-cadherin regulation in cancers in human beings. Furthermore, the aggressive nature of xenografts positive for E-cadherin and the frequency of metastases positive for E-cadherin suggest that high E-cadherin expression in metastatic prostate cancer is associated with aggressive tumor growth.

Characterization of adipose-derived stem cells: an update.

Adipose tissue is an attractive source of multipotent adult stem cells due to its wide-spread availability, accessibility, and ease of harvest. Adipose-derived stem cells (ASCs), the adherent stromal cell population present within adipose tissue, are easily expanded in culture, able to differentiate along multiple cell-lineage pathways, and have been shown to provide therapeutic benefit in models of injury and disease through immunomodulation, structural integation, and/or trophic support. Recent developments in the characterization of ASCs, specifically their isolation, gene and protein expression, differentiation, and expansion, are reviewed in this article.

Agent-based model of therapeutic adipose-derived stromal cell trafficking during ischemia predicts ability to roll on P-selectin.

Intravenous delivery of human adipose-derived stromal cells (hASCs) is a promising option for the treatment of ischemia. After delivery, hASCs that reside and persist in the injured extravascular space have been shown to aid recovery of tissue perfusion and function, although low rates of incorporation currently limit the safety and efficacy of these therapies. We submit that a better understanding of the trafficking of therapeutic hASCs through the microcirculation is needed to address this and that selective control over their homing (organ- and injury-specific) may be possible by targeting bottlenecks in the homing process. This process, however, is incredibly complex, which merited the use of computational techniques to speed the rate of discovery. We developed a multicell agent-based model (ABM) of hASC trafficking during acute skeletal muscle ischemia, based on over 150 literature-based rules instituted in Netlogo and MatLab software programs. In silico, trafficking phenomena within cell populations emerged as a result of the dynamic interactions between adhesion molecule expression, chemokine secretion, integrin affinity states, hemodynamics and microvascular network architectures. As verification, the model reasonably reproduced key aspects of ischemia and trafficking behavior including increases in wall shear stress, upregulation of key cellular adhesion molecules expressed on injured endothelium, increased secretion of inflammatory chemokines and cytokines, quantified levels of monocyte extravasation in selectin knockouts, and circulating monocyte rolling distances. Successful ABM verification prompted us to conduct a series of systematic knockouts in silico aimed at identifying the most critical parameters mediating hASC trafficking. Simulations predicted the necessity of an unknown selectin-binding molecule to achieve hASC extravasation, in addition to any rolling behavior mediated by hASC surface expression of CD15s, CD34, CD62e, CD62p, or CD65. In vitro experiments confirmed this prediction; a subpopulation of hASCs slowly rolled on immobilized P-selectin at speeds as low as 2 microm/s. Thus, our work led to a fundamentally new understanding of hASC biology, which may have important therapeutic implications.

Arteriolar remodeling following ischemic injury extends from capillary to large arteriole in the microcirculation.

OBJECTIVE: Skeletal muscle vasculature undergoes arteriogenesis to restore tissue perfusion and function following loss of blood flow. This process has been shown to occur in large vessels following ischemia, although recent studies suggest this may occur in the microcirculation as well. We tested the hypothesis that ischemia induces microvascular remodeling in the skeletal muscle microcirculation on the scale of capillary to sub-35 mum diameter arterioles. METHODS: Ligations of a feeding arteriole to the caudal-half of the spinotrapezius muscle were performed on C57BL/6 mice. At 5 days, microvascular remodeling responses were quantified using intravital and whole-mount confocal microscopy. Immunohistochemistry was performed to visualize vessels, incorporated leukocytes, and regions of hypoxia. RESULTS: Ischemic tissue underwent localized microvascular remodeling characteristic of arteriogenesis, including pronounced vessel tortuosity. In patent microvessels (diameters 15-35 microm), we observed increases in vascular density (38%), branching (90%) and collateral development (36.5%). The formation of new arterioles (diameters 6-35 microm) increased by 24.3%, while chronic hypoxia was absent from all tissues. CONCLUSIONS: Ischemic injury induces arteriogenesis in skeletal muscle microcirculation. Furthermore, this surgical model enables en face analysis of microcirculatory adaptations with single-cell resolution and can provide investigators with morphometric data on a microscale that is difficult to achieve using other models.

IFATS collection: The role of human adipose-derived stromal cells in inflammatory microvascular remodeling and evidence of a perivascular phenotype.

A growing body of literature suggests that human adipose-derived stromal cells (hASCs) possess developmental plasticity both in vitro and in vivo, and might represent a viable cell source for therapeutic angiogenesis and tissue engineering. We investigate their phenotypic similarity to perivascular cell types, ability to contribute to in vivo microvascular remodeling, and ability to modulate vascular stability. We evaluated hASC surface expression of vascular and stem/progenitor cell markers in vitro, as well as any effects of platelet-derived growth factor B chain (PDGF-BB) and vascular endothelial growth factor 165 on in vitro hASC migration. To ascertain in vivo behavior of hASCs in an angiogenic environment, hASCs were isolated, expanded in culture, labeled with a fluorescent marker, and injected into adult nude rat mesenteries that were stimulated to undergo microvascular remodeling. Ten, 30, and 60 days after injection, tissues from anesthetized animals were harvested and processed with immunohistochemical techniques to determine hASC quantity, positional fate in relation to microvessels, and expression of endothelial and perivascular cell markers. After 60 days, 29% of hASCs exhibited perivascular morphologies compared with 11% of injected human lung fibroblasts. hASCs exhibiting perivascular morphologies also expressed markers characteristic of vascular pericytes: smooth muscle alpha-actin (10%) and neuron-glia antigen 2 (8%). In tissues treated with hASCs, vascular density was significantly increased over age-matched controls lacking hASCs. This study demonstrates that hASCs express pericyte lineage markers in vivo and in vitro, exhibit increased migration in response to PDGF-BB in vitro, exhibit perivascular morphology when injected in vivo, and contribute to increases in microvascular density during angiogenesis by migrating toward vessels. Disclosure of potential conflicts of interest is found at the end of this article.

Functional binding of human adipose-derived stromal cells: effects of extraction method and hypoxia pretreatment.

Human adipose-derived stromal cells (hASCs) were evaluated in vitro for their ability to bind vascular adhesion and extracellular matrix proteins to arrest (firmly adhere) under physiological flow conditions. hASCs were flowed through a parallel plate flow chamber containing substrates presenting immobilized type I collagen, fibronectin, E-selectin, L-selectin, P-selectin, vascular cell adhesion molecule-1 (VCAM-1), or intercellular adhesion molecule-1 (ICAM-1) under static and laminar flow conditions (wall shear stress = 1 dyn/cm). hASCs were able to firmly adhere to type I collagen, fibronectin, VCAM-1, and ICAM-1 substrates, but not to any of the selectins. Pretreatment with hypoxia increased the ability of hASCs isolated by liposuction to adhere to VCAM-1 and ICAM-1, but this effect was not seen in cells isolated by tissue excision. These results indicate that hASCs possess the ability to adhere key adhesion proteins, illustrate the importance of hASC harvest procedure, and suggest mechanisms for homing in a setting where interaction with inflamed or injured tissue is necessary.