Pharmacological Inhibition of PTEN Rescues Dopaminergic Neurons by Attenuating Apoptotic and Neuroinflammatory Signaling Events.

Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by the selective degeneration of dopaminergic neurons in the substantia nigra pars compacta resulting in an irreversible and a debilitating motor dysfunction. Though both genetic and idiopathic factors are implicated in the disease etiology, idiopathic PD comprise the majority of clinical cases and is caused by exposure to environmental toxicants and oxidative stress. Fyn kinase activation has been identified as an early molecular signaling event that primes neuroinflammatory and neurodegenerative events associated with dopaminergic cell death. However, the upstream regulator of Fyn activation remains unidentified. We investigated whether the lipid and tyrosine phosphatase PTEN (Phosphatase and Tensin homolog deleted on chromosome 10) could be the upstream regulator of Fyn activation in PD models as PTEN has been previously reported to contribute to Parkinsonian pathology. Our findings, using bioluminescence resonance energy transfer (BRET) and immunoblotting, indicate for the first time that PTEN is a critical early stress sensor in response to oxidative stress and neurotoxicants in in vitro models of PD. Pharmacological attenuation of PTEN activity rescues dopaminergic neurons from neurotoxicant-induced cytotoxicity by modulating Fyn kinase activation. Our findings also identify PTEN's novel roles in contributing to mitochondrial dysfunction which contribute to neurodegenerative processes. Interestingly, we found that PTEN positively regulates interleukin-1ß (IL-1ß) and the transcription of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Taken together, we have identified PTEN as a disease course altering pharmacological target that may be further validated for the development of novel therapeutic strategies targeting PD.

Does lower extremity fracture fixation technique influence neurologic outcomes in patients with traumatic brain injury? The EAST Brain vs. Bone multicenter trial.

OBJECTIVE: This study aimed to determine whether lower extremity fracture fixation technique and timing (≤24 vs. >24 hours) impact neurologic outcomes in TBI patients. METHODS: A prospective observational study was conducted across 30 trauma centers. Inclusion criteria were age 18 years and older, head Abbreviated Injury Scale (AIS) score of >2, and a diaphyseal femur or tibia fracture requiring external fixation (Ex-Fix), intramedullary nailing (IMN), or open reduction and internal fixation (ORIF). The analysis was conducted using analysis of variamce, Kruskal-Wallis, and multivariable regression models. Neurologic outcomes were measured by discharge Ranchos Los Amigos Revised Scale (RLAS-R). RESULTS: Of the 520 patients enrolled, 358 underwent Ex-Fix, IMN, or ORIF as definitive management. Head AIS was similar among cohorts. The Ex-Fix group experienced more severe lower extremity injuries (AIS score, 4-5) compared with the IMN group (16% vs. 3%, p = 0.01) but not the ORIF group (16% vs. 6%, p = 0.1). Time to operative intervention varied between the cohorts with the longest time to intervention for the IMN group (median hours: Ex-Fix, 15 [8-24] vs. ORIF, 26 [12-85] vs. IMN, 31 [12-70]; p < 0.001). The discharge RLAS-R score distribution was similar across the groups. After adjusting for confounders, neither method nor timing of lower extremity fixation influenced the discharge RLAS-R. Instead, increasing age and head AIS score were associated with a lower discharge RLAS-R score (odds ratio [OR], 1.02; 95% confidence interval [CI], 1.002-1.03 and OR, 2.37; 95% CI, 1.75-3.22), and a higher Glasgow Coma Scale motor score on admission (OR, 0.84; 95% CI, 0.73-0.97) was associated with higher RLAS-R score at discharge. CONCLUSION: Neurologic outcomes in TBI are impacted by severity of the head injury and not the fracture fixation technique or timing. Therefore, the strategy of definitive fixation of lower extremity fractures should be dictated by patient physiology and the anatomy of the injured extremity and not by the concern for worsening neurologic outcomes in TBI patients. LEVEL OF EVIDENCE: Prognostic and Epidemiological; Level III.

Pharmacological chaperone-rescued cystic fibrosis CFTR-F508del mutant overcomes PRAF2-gated access to endoplasmic reticulum exit sites.

The endoplasmic reticulum exit of some polytopic plasma membrane proteins (PMPs) is controlled by arginin-based retention motifs. PRAF2, a gatekeeper which recognizes these motifs, was shown to retain the GABAB-receptor GB1 subunit in the ER. We report that PRAF2 can interact on a stoichiometric basis with both wild type and mutant F508del Cystic Fibrosis (CF) Transmembrane Conductance Regulator (CFTR), preventing the access of newly synthesized cargo to ER exit sites. Because of its lower abundance, compared to wild-type CFTR, CFTR-F508del recruitment into COPII vesicles is suppressed by the ER-resident PRAF2. We also demonstrate that some pharmacological chaperones that efficiently rescue CFTR-F508del loss of function in CF patients target CFTR-F508del retention by PRAF2 operating with various mechanisms. Our findings open new therapeutic perspectives for diseases caused by the impaired cell surface trafficking of mutant PMPs, which contain RXR-based retention motifs that might be recognized by PRAF2.

Differential Leukocyte MicroRNA Responses Following Pan T Cell, Allorecognition and Allosecretome-Based Therapeutic Activation.

Effective immunomodulation of T-cell responses is critical in treating both autoimmune diseases and cancer. Our previous studies have demonstrated that secretomes derived from control or methoxypolyethylene glycol mixed lymphocyte alloactivation assays exerted potent immunomodulatory activity that was mediated by microRNAs (miRNA). The immunomodulatory effects of biomanufactured miRNA-based allo-secretome therapeutics (SYN, TA1, IA1 and IA2) were compared to Pan T-cell activators (PHA and anti-CD3/CD28) and lymphocyte alloactivation. The differential effects of these activation strategies on resting peripheral blood mononuclear cells (PBMC) were assessed via T-cell proliferation, subset analysis and miRNA expression profiles. Mitogen-induced PBMC proliferation (> 85%) significantly exceeded that arising from either allostimulation (~ 30%) or the pro-inflammatory IA1 secretome product (~ 12%). Consequent to stimulation, the ratio of CD4 to CD8 cells of the resting PBMC (CD4:CD8; 1.7 ± 0.1) decreased in the Pan T cell, allrecognition and IA1 activated cells (averages of 1.1 ± 0.2; 1.2 ± 0.1 and 1.0 ± 0.1). These changes arose consequent to the expansion of both CD4+CD8+ and CD4-CD8- populations as well as the shrinkage of the CD4 subset and the expansion of the CD8 T cells. Importantly, these activation strategies induced vastly different miRNA expression profiles which were associated with significant differences in cellular differentiation and biological function. These findings support the concept that the "differential patterns of miRNA expression" regulate the biologic immune response in a "lock and key" manner. The biomanufacturing of miRNA-enriched secretome biotherapeutics may be a successful therapeutic approach for the systemic treatment of autoimmune diseases (TA1) and cancer (IA1).

Live-imaging of endothelial Erk activity reveals dynamic and sequential signalling events during regenerative angiogenesis.

The formation of new blood vessel networks occurs via angiogenesis during development, tissue repair, and disease. Angiogenesis is regulated by intracellular endothelial signalling pathways, induced downstream of vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs). A major challenge in understanding angiogenesis is interpreting how signalling events occur dynamically within endothelial cell populations during sprouting, proliferation, and migration. Extracellular signal-regulated kinase (Erk) is a central downstream effector of Vegf-signalling and reports the signalling that drives angiogenesis. We generated a vascular Erk biosensor transgenic line in zebrafish using a kinase translocation reporter that allows live-imaging of Erk-signalling dynamics. We demonstrate the utility of this line to live-image Erk activity during physiologically relevant angiogenic events. Further, we reveal dynamic and sequential endothelial cell Erk-signalling events following blood vessel wounding. Initial signalling is dependent upon Ca2+ in the earliest responding endothelial cells, but is independent of Vegfr-signalling and local inflammation. The sustained regenerative response, however, involves a Vegfr-dependent mechanism that initiates concomitantly with the wound inflammatory response. This work reveals a highly dynamic sequence of signalling events in regenerative angiogenesis and validates a new resource for the study of vascular Erk-signalling in real-time.

The RanBP2/RanGAP1-SUMO complex gates ß-arrestin2 nuclear entry to regulate the Mdm2-p53 signaling axis.

Mdm2 antagonizes the tumor suppressor p53. Targeting the Mdm2-p53 interaction represents an attractive approach for the treatment of cancers with functional p53. Investigating mechanisms underlying Mdm2-p53 regulation is therefore important. The scaffold protein ß-arrestin2 (ß-arr2) regulates tumor suppressor p53 by counteracting Mdm2. ß-arr2 nucleocytoplasmic shuttling displaces Mdm2 from the nucleus to the cytoplasm resulting in enhanced p53 signaling. ß-arr2 is constitutively exported from the nucleus, via a nuclear export signal, but mechanisms regulating its nuclear entry are not completely elucidated. ß-arr2 can be SUMOylated, but no information is available on how SUMO may regulate ß-arr2 nucleocytoplasmic shuttling. While we found ß-arr2 SUMOylation to be dispensable for nuclear import, we identified a non-covalent interaction between SUMO and ß-arr2, via a SUMO interaction motif (SIM), that is required for ß-arr2 cytonuclear trafficking. This SIM promotes association of ß-arr2 with the multimolecular RanBP2/RanGAP1-SUMO nucleocytoplasmic transport hub that resides on the cytoplasmic filaments of the nuclear pore complex. Depletion of RanBP2/RanGAP1-SUMO levels result in defective ß-arr2 nuclear entry. Mutation of the SIM inhibits ß-arr2 nuclear import, its ability to delocalize Mdm2 from the nucleus to the cytoplasm and enhanced p53 signaling in lung and breast tumor cell lines. Thus, a ß-arr2 SIM nuclear entry checkpoint, coupled with active ß-arr2 nuclear export, regulates its cytonuclear trafficking function to control the Mdm2-p53 signaling axis.

TRPV1 Activation Promotes ß-arrestin2 Interaction with the Ribosomal Biogenesis Machinery in the Nucleolus:Implications for p53 Regulation and Neurite Outgrowth.

Transient receptor potential vanilloids (TRPV1) are non-selective cation channels that sense and transduce inflammatory pain signals. We previously reported that activation of TRPV1 induced the translocation of ß-arrestin2 (ARRB2) from the cytoplasm to the nucleus, raising questions about the functional role of ARRB2 in the nucleus. Here, we determined the ARRB2 nuclear signalosome by conducting a quantitative proteomic analysis of the nucleus-sequestered L395Q ARRB2 mutant, compared to the cytosolic wild-type ARRB2 (WT ARRB2), in a heterologous expression system. We identified clusters of proteins that localize to the nucleolus and are involved in ribosomal biogenesis. Accordingly, L395Q ARRB2 or WT ARRB2 after capsaicin treatment were found to co-localize and interact with the nucleolar marker nucleophosmin (NPM1), treacle protein (TCOF1) and RNA polymerase I (POL I). We further investigated the role of nuclear ARRB2 signaling in regulating neuroplasticity. Using neuroblastoma (neuro2a) cells and dorsal root ganglia (DRG) neurons, we found that L395Q ARRB2 mutant increased POL I activity, inhibited the tumor suppressorp53 (p53) level and caused a decrease in the outgrowth of neurites. Together, our results suggest that the activation of TRPV1 promotes the ARRB2-mediated regulation of ribosomal biogenesis in the nucleolus. The ARRB2-TCOF1-p53 checkpoint signaling pathway might be involved in regulating neurite outgrowth associated with pathological pain conditions.

Discovery of novel small molecule inhibitors of S100P with in vitro anti-metastatic effects on pancreatic cancer cells.

S100P, a calcium-binding protein, is known to advance tumor progression and metastasis in pancreatic and several other cancers. Herein is described the in silico identification of a putative binding pocket of S100P to identify, synthesize and evaluate novel small molecules with the potential to selectively bind S100P and inhibit its activation of cell survival and metastatic pathways. The virtual screening of a drug-like database against the S100P model led to the identification of over 100 clusters of diverse scaffolds. A representative test set identified a number of structurally unrelated hits that inhibit S100P-RAGE interaction, measured by ELISA, and reduce in vitro cell invasion selectively in S100P-expressing pancreatic cancer cells at 10 µM. This study establishes a proof of concept in the potential for rational design of small molecule S100P inhibitors for drug candidate development.

Beta-arrestins operate an on/off control switch for focal adhesion kinase activity.

Focal adhesion kinase (FAK) regulates key biological processes downstream of G protein-coupled receptors (GPCRs) in normal and cancer cells, but the modes of kinase activation by these receptors remain unclear. We report that after GPCR stimulation, FAK activation is controlled by a sequence of events depending on the scaffolding proteins ß-arrestins and G proteins. Depletion of ß-arrestins results in a marked increase in FAK autophosphorylation and focal adhesion number. We demonstrate that ß-arrestins interact directly with FAK and inhibit its autophosphorylation in resting cells. Both FAK-ß-arrestin interaction and FAK inhibition require the FERM domain of FAK. Following the stimulation of the angiotensin receptor AT1AR and subsequent translocation of the FAK-ß-arrestin complex to the plasma membrane, ß-arrestin interaction with the adaptor AP-2 releases inactive FAK from the inhibitory complex, allowing its activation by receptor-stimulated G proteins and activation of downstream FAK effectors. Release and activation of FAK in response to angiotensin are prevented by an AP-2-binding deficient ß-arrestin and by a specific inhibitor of ß-arrestin/AP-2 interaction; this inhibitor also prevents FAK activation in response to vasopressin. This previously unrecognized mechanism of FAK regulation involving a dual role of ß-arrestins, which inhibit FAK in resting cells while driving its activation at the plasma membrane by GPCR-stimulated G proteins, opens new potential therapeutic perspectives in cancers with up-regulated FAK.

Posttranslational Regulation and Conformational Plasticity of PTEN.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a tumor suppressor that is frequently down-modulated in human cancer. PTEN inhibits the phosphatidylinositol 3-phosphate kinase (PI3K)/AKT pathway through its lipid phosphatase activity. Multiple PI3K/AKT-independent actions of PTEN, protein-phosphatase activities and functions within the nucleus have also been described. PTEN, therefore, regulates many cellular processes including cell proliferation, survival, genomic integrity, polarity, migration, and invasion. Even a modest decrease in the functional dose of PTEN may promote cancer development. Understanding the molecular and cellular mechanisms that regulate PTEN protein levels and function, and how these may go awry in cancer contexts, is, therefore, key to fully understanding the role of PTEN in tumorigenesis. Here, we discuss current knowledge on posttranslational control and conformational plasticity of PTEN, as well as therapeutic possibilities toward reestablishment of PTEN tumor-suppressor activity in cancer.

Trends and characteristics of cases when serial carboxyhemoglobins are obtained.

BACKGROUND: Carboxyhemoglobin (COHb) levels are obtained when there is suspicion for carbon monoxide (CO) exposure. Serial COHb levels are sometimes obtained despite the well-established half-life of COHb with oxygen supplementation. We sought to evaluate the trends and characteristics associated with obtaining serial carboxyhemoglobin levels. METHODS: A retrospective review was performed at an academic medical center for all inpatient and emergency department cases with either single COHb or serial COHb levels from 1 April 2010 through 31 March 2015. Data collected included age, gender, pregnancy status, smoking history, encounter month, admission status, oxygen administration, fire or burn history, vital signs, presenting symptoms, hyperbaric oxygen (HBO2) therapy use, initial pH, troponin, lactate, and COHb levels. The time and change in values between serial levels were also obtained. RESULTS: 624 cases were identified, with 106 (17%) having multiple carboxyhemoglobin levels. A mean of 2.6 (range 2 - 9) serial COHb levels were obtained. The average initial COHb was 8.9%. Subsequent serial levels were obtained on average at 353, 663 and 1,095 minutes and averaged 2.8%, 1.8% and 1.1% respectively. Serial COHb levels were obtained more commonly in burn patients, those admitted to the ICU and those who had HBO2 therapy. Four patients had an increase in COHb level on serial testing. The largest increase of these was from 2.0% to 3.9%. CONCLUSION: Serial COHb levels were not infrequent in this study. No clinically significant increase in COHb was identified by serial testing. Further studies should examine the clinical utility of such practices.

Biomarkers of oral inflammation in perinatally HIV-infected and perinatally HIV-exposed, uninfected youth.

AIM: To examine oral biomarkers that have been associated with periodontal disease progression in HIV-infected adults in perinatally HIV-infected and HIV-exposed but uninfected youth. MATERIAL AND METHODS: This was a cross-sectional, multicentre substudy of youth participating in the Oral Health Pediatric HIV/AIDS Cohort study. Gingival crevicular fluid repository samples from participants with and without periodontal disease (using Gingival Index [GI] and Bleeding on Probing [BOP] parameters on dental examination) were tested for concentration levels of inflammatory biomarkers. Associations were assessed using Wilcoxon test and Spearman correlation. RESULTS: For perinatal HIV youth (n = 129), the markers consistently elevated (p < .05) in sites with GI ≥2 and in sites with BOP were interleukin-1ß, 6 and 13, macrophage inflammatory protein-1α and metalloproteinase-9. Serum tumour necrosis factor-α and soluble CD14 were positively correlated with a summary count of elevated cytokines. No associations were seen among HIV-uninfected subjects (n = 71). CONCLUSIONS: The association of oral biomarkers of inflammation with clinical indicators of periodontal inflammation and systemic immune activation suggests that perinatal HIV-infected youth may be at higher risk for developing significant periodontal disease, associated with tooth loss and HIV progression. More frequent dental care of this group is needed to prevent potential periodontal progression.

Identification of Kinases Responsible for p53-Dependent Autophagy.

In cancer, autophagy is upregulated to promote cell survival and tumor growth during times of nutrient stress and can confer resistance to drug treatments. Several major signaling networks control autophagy induction, including the p53 tumor suppressor pathway. In response to DNA damage and other cellular stresses, p53 is stabilized and activated, while HDM2 binds to and ubiquitinates p53 for proteasome degradation. Thus blocking the HDM2-p53 interaction is a promising therapeutic strategy in cancer; however, the potential survival advantage conferred by autophagy induction may limit therapeutic efficacy. In this study, we leveraged an HDM2 inhibitor to identify kinases required for p53-dependent autophagy. Interestingly, we discovered that p53-dependent autophagy requires several kinases, including the myotonic dystrophy protein kinase-like alpha (MRCKα). MRCKα is a CDC42 effector reported to activate actin-myosin cytoskeletal reorganization. Overall, this study provides evidence linking MRCKα to autophagy and reveals additional insights into the role of kinases in p53-dependent autophagy.

Biocompatibility and toxicity of novel iron chelator Starch-Deferoxamine (S-DFO) compared to zinc oxide nanoparticles to zebrafish embryo: An oxidative stress based apoptosis, physicochemical and neurological study profile.

OBJECTIVES: Clinically approved iron chelators are effective in decreasing significant transfusional iron accumulation. Starch-Deferoxamine (S-DFO), a novel high molecular weight iron chelator, was produced to increase binding capacity to iron and reduce toxicity. Although its efficacy was established in one small cohort clinical trial, its potential adverse effect was not adequately addressed. METHODS: We utilized zebrafish model to assess S-DFO toxicity using following assays: mortality, teratogenicity, hatching rate, tail flicking, Acridine Orange staining for apoptosis detection, o-dianisidine staining for hemoglobin synthesis, and the level of Hsp70 as a general stress indicator. Embryos were exposed to different concentrations of S-DFO, Zinc Oxide nanoparticle (ZnO) (positive control), along with untreated control (UC). RESULTS: S-DFO showed no significant mortality nor deformities at all tested concentrations (0.0-1000 µM). Thus, the LC50 is expected to >1000 µM. 100 µM S-DFO treatment did not affect embryo development (as judged by hatching rate); neuromuscular activity (as judged by tail flicking); and hemoglobin synthesis. Neither apoptosis, nor increase in Hsp70 level was noticed upon S-DFO treatment. CONCLUSION: Our assays demonstrate that S-DFO does not induce cellular or biochemical stress and has no adverse effect on organ development of zebrafish embryos, suggesting its safe use as an iron chelator.

Enhancing the pro-inflammatory anti-cancer T cell response via biomanufactured, secretome-based, immunotherapeutics.

T lymphocytes play a critical role in the pro-inflammatory anti-cancer response; hence, significant pharmacologic efforts have been made to enhance the endogenous T cell response. Unfortunately, significant toxicity arises consequent to pan T cell activation. In contrast, the less robust T cell alloresponse has also demonstrated an anti-cancer effect, but poses an inherent risk of GvHD. To overcome the GvHD risk, an acellular pro-inflammatory agent (IA1) has been biomanufactured from the secretome of the allorecognition response. To assess IA1's immunomodulatory activity, T cell proliferation and differentiation were determined in vitro. The pro-inflammatory properties of the IA1 therapeutic were mediated by the miRNA-enriched fractions. Moreover, cross-species efficacy was observed consequent to the evolutionary conservation of miRNA. IA1 exerted no toxicity to resting PBMC but induced significant proliferation of resting CD3+ (CD4+ and CD8+) T cells and skewed the response towards a pro-inflammatory state (i.e., increased Teff:Treg ratio). Crucially, IA1-activated PBMC demonstrated a potent inhibition of cancer cell (HeLa and SH-4 melanoma) proliferation relative to the resting PBMC. The anti-proliferation effect of IA1-activated PBMC was noted within ˜12 h versus 4-5 days for resting cells. A second biomanufactured therapeutic (IA2; produced using HeLa cells) surprisingly demonstrated direct toxicity to cancer cells but was less effective than IA1 in inducing a cell-mediated response. This study demonstrates that miRNA-enriched therapeutics can be biomanufactured from the secretome and can induce a potent pro-inflammatory, anti-cancer, effect on resting lymphocytes.

Skin complications associated with vascular access devices: A secondary analysis of 13 studies involving 10,859 devices.

BACKGROUND: Vascular access devices are widely used in healthcare settings worldwide. The insertion of a vascular access device creates a wound, vulnerable to irritation, injury and infection. Vascular access-associated skin complications are frequently reported in the literature, however very little evidence is available regarding the incidence and risk factors of these conditions to inform practice and technology development. OBJECTIVES: To estimate the incidence of vascular access-associated skin complications, and to identify patient, catheter and healthcare-related characteristics associated with skin complication development. DESIGN: Secondary data analysis from 13 multi-centre randomised controlled trials and observational studies evaluating technologies and performance of vascular access devices in clinical settings between 2008 and 2017. SETTINGS: Six hospitals (metropolitan and regional) in Queensland, Australia. PARTICIPANTS: The 13 studies involved paediatric and adult participants, across oncology, emergency, intensive care, and general hospital settings. A total of 7669 participants with 10,859 devices were included, involving peripheral venous (n = 9933), peripheral arterial (n = 341), and central venous access (n = 585) devices. ANALYSIS: Standardised study data were extracted into a single database. Clinical and demographic data were descriptively reported. Cox proportional hazards regression models (stratified by peripheral vs central) were used for time-to-event, per-device analyses to examine risk factors. Univariate associations were undertaken due to complexities with missing data in both outcomes and covariates, with p < 0.01 to reduce the effect of multiple comparisons. RESULTS: Over 12% of devices were associated with skin complication, at 46.2 per 1000 catheter days for peripheral venous and arterial devices (95% confidence interval, CI 42.1-50.7), and 22.5 per 1000 catheter days for central devices (95% CI 16.5-306). The most common skin complications were bruising (peripheral n = 134, 3.7%; central n = 33, 6.8%), and swelling due to infiltration for peripheral devices (n = 296; 2.9%), and dermatitis for central devices (n = 13; 2.2%). The significant risk factors for these complications were predominantly related to device (e.g., skin tears associated with peripheral arterial catheters [hazard ratio, HR 16.0], radial insertion [HR 18.0] basilic insertion [HR 26.0])) and patient characteristics (e.g., poor skin integrity associated with increased risk of peripheral device bruising [HR 4.12], infiltration [HR 1.98], and skin tear [HR 48.4]), rather than management approaches. CONCLUSIONS: Significant skin complications can develop during the life of peripheral and central vascular access devices, and these are associated with several modifiable and non-modifiable risk factors. Further research is needed to evaluate effectiveness technologies to prevent and treat skin complications associated with vascular access devices.

Imaging methods used to study mouse and human HSC niches: Current and emerging technologies.

Bone marrow contains numerous different cell types arising from hematopoietic stem cells (HSCs) and non-hematopoietic mesenchymal/skeletal stem cells, in addition to other cell types such as endothelial cells- these non-hematopoietic cells are commonly referred to as stromal cells or microenvironment cells. HSC function is intimately linked to complex signals integrated by their niches, formed by combinations of hematopoietic and stromal cells. Studies of hematopoietic cells have been significantly advanced by flow cytometry methods, enabling the quantitation of each cell type in normal and perturbed situations, in addition to the isolation of these cells for molecular and functional studies. Less is known, however, about the specific niches for distinct developing hematopoietic lineages, or the changes occurring in the niche size and function in these distinct anatomical sites in the bone marrow under stress situations and ageing. Significant advances in imaging technology during the last decade have permitted studies of HSC niches in mice. Additional imaging technologies are emerging that will facilitate the study of human HSC niches in trephine BM biopsies. Here we provide an overview of imaging technologies used to study HSC niches, in addition to highlighting emerging technology that will help us to more precisely identify and characterize HSC niches in normal and diseased states.

A Potent and Selective ULK1 Inhibitor Suppresses Autophagy and Sensitizes Cancer Cells to Nutrient Stress.

In response to stress, cancer cells generate nutrients and energy through a cellular recycling process called autophagy, which can promote survival and tumor progression. Accordingly, autophagy inhibition has emerged as a potential cancer treatment strategy. Inhibitors targeting ULK1, an essential and early autophagy regulator, have provided proof of concept for targeting this kinase to inhibit autophagy; however, these are limited individually in their potency, selectivity, or cellular activity. In this study, we report two small molecule ULK1 inhibitors, ULK-100 and ULK-101, and establish superior potency and selectivity over a noteworthy published inhibitor. Moreover, we show that ULK-101 suppresses autophagy induction and autophagic flux in response to different stimuli. Finally, we use ULK-101 to demonstrate that ULK1 inhibition sensitizes KRAS mutant lung cancer cells to nutrient stress. ULK-101 represents a powerful molecular tool to study the role of autophagy in cancer cells and to evaluate the therapeutic potential of autophagy inhibition.

Subtle Changes in the Levels of BCL-2 Proteins Cause Severe Craniofacial Abnormalities.

Apoptotic cell death removes unwanted cells and is regulated by interactions between pro-survival and pro-apoptotic members of the BCL-2 protein family. The regulation of apoptosis is thought to be crucial for normal embryonic development. Accordingly, complete loss of pro-survival MCL-1 or BCL-XL (BCL2L1) causes embryonic lethality. However, it is not known whether minor reductions in pro-survival proteins could cause developmental abnormalities. We explored the rate-limiting roles of MCL-1 and BCL-XL in development and show that combined loss of single alleles of Mcl-1 and Bcl-x causes neonatal lethality. Mcl-1+/-;Bcl-x+/- mice display craniofacial anomalies, but additional loss of a single allele of pro-apoptotic Bim (Bcl2l11) restores normal development. These findings demonstrate that the control of cell survival during embryogenesis is finely balanced and suggest that some human craniofacial defects, for which causes are currently unknown, may be due to subtle imbalances between pro-survival and pro-apoptotic BCL-2 family members.