Metabolic vulnerability of cancer stem cells and their niche.

Cancer stem cells (CSC) are the leading cause of the failure of anti-tumor treatments. These aggressive cancer cells are preserved and sustained by adjacent cells forming a specialized microenvironment, termed niche, among which tumor-associated macrophages (TAMs) are critical players. The cycle of tricarboxylic acids, fatty acid oxidation path, and electron transport chain have been proven to play central roles in the development and maintenance of CSCs and TAMs. By improving their oxidative metabolism, cancer cells are able to extract more energy from nutrients, which allows them to survive in nutritionally defective environments. Because mitochondria are crucial bioenergetic hubs and sites of these metabolic pathways, major hopes are posed for drugs targeting mitochondria. A wide range of medications targeting mitochondria, electron transport chain complexes, or oxidative enzymes are currently investigated in phase 1 and phase 2 clinical trials against hard-to-treat tumors. This review article aims to highlight recent literature on the metabolic adaptations of CSCs and their supporting macrophages. A focus is provided on the resistance and dormancy behaviors that give CSCs a selection advantage and quiescence capacity in particularly hostile microenvironments and the role of TAMs in supporting these attitudes. The article also describes medicaments that have demonstrated a robust ability to disrupt core oxidative metabolism in preclinical cancer studies and are currently being tested in clinical trials.

The FKBP51s Splice Isoform Predicts Unfavorable Prognosis in Patients with Glioblastoma.

The primary treatment for glioblastoma (GBM) is removing the tumor mass as defined by MRI. However, MRI has limited diagnostic and predictive value. Tumor-associated macrophages (TAM) are abundant in GBM tumor microenvironment (TME) and are found in peripheral blood (PB). FKBP51 expression, with its canonical and spliced isoforms, is constitutive in immune cells and aberrant in GBM. Spliced FKBP51s supports M2 polarization. To find an immunologic signature that combined with MRI could advance in diagnosis, we immunophenotyped the macrophages of TME and PB from 37 patients with GBM using FKBP51s and classical M1-M2 markers. We also determined the tumor levels of FKBP51s, PD-L1, and HLA-DR. Tumors expressing FKBP51s showed an increase in various M2 phenotypes and regulatory T cells in PB, indicating immunosuppression. Tumors expressing FKBP51s also activated STAT3 and were associated with reduced survival. Correlative studies with MRI and tumor/macrophages cocultures allowed to interpret TAMs. Tumor volume correlated with M1 infiltration of TME. Cocultures with spheroids produced M1 polarization, suggesting that M1 macrophages may infiltrate alongside cancer stem cells. Cocultures of adherent cells developed the M2 phenotype CD163/FKBP51s expressing pSTAT6, a transcription factor enabling migration and invasion. In patients with recurrences, increased counts of CD163/FKBP51s monocyte/macrophages in PB correlated with callosal infiltration and were accompanied by a concomitant decrease in TME-infiltrating M1 macrophages. PB PD-L1/FKBP51s connoted necrotic tumors. In conclusion, FKBP51s identifies a GBM subtype that significantly impairs the immune system. Moreover, FKBP51s marks PB macrophages associated with MRI features of glioma malignancy that can aid in patient monitoring. SIGNIFICANCE: Our research suggests that by combining imaging with analysis of monocyte/macrophage subsets in patients with GBM, we can enhance our understanding of the disease and assist in its treatment. We discovered a similarity in the macrophage composition between the TME and PB, and through association with imaging, we could interpret macrophages. In addition, we identified a predictive biomarker that drew more attention to immune suppression of patients with GBM.

Scaffold proteins of cancer signaling networks: The paradigm of FK506 binding protein 51 (FKBP51) supporting tumor intrinsic properties and immune escape.

Scaffold proteins are crucial regulators of signaling networks, and their abnormal expression may favor the development of tumors. Among the scaffold proteins, immunophilin covers a unique role as 'protein-philin' (Greek 'philin' = friend) that interacts with proteins to guide their proper assembly. The growing list of human syndromes associated with the immunophilin defect underscores the biological relevance of these proteins that are largely opportunistically exploited by cancer cells to support and enable the tumor's intrinsic properties. Among the members of the immunophilin family, the FKBP5 gene was the only one identified to have a splicing variant. Cancer cells impose unique demands on the splicing machinery, thus acquiring a particular susceptibility to splicing inhibitors. This review article aims to overview the current knowledge of the FKBP5 gene functions in human cancer, illustrating how cancer cells exploit the scaffolding function of canonical FKBP51 to foster signaling networks that support their intrinsic tumor properties and the spliced FKBP51s to gain the capacity to evade the immune system.

FKBP51 plays an essential role in Akt ubiquitination that requires Hsp90 and PHLPP.

FKBP51 plays a relevant role in sustaining cancer cells, particularly melanoma. This cochaperone participates in several signaling pathways. FKBP51 forms a complex with Akt and PHLPP, which is reported to dephosphorylate Akt. Given the recent discovery of a spliced FKBP51 isoform, in this paper, we interrogate the canonical and spliced isoforms in regulation of Akt activation. We show that the TPR domain of FKBP51 mediates Akt ubiquitination at K63, which is an essential step for Akt activation. The spliced FKBP51, lacking such domain, cannot link K63-Ub residues to Akt. Unexpectedly, PHLPP silencing does not foster phosphorylation of Akt, and its overexpression even induces phosphorylation of Akt. PHLPP stabilizes levels of E3-ubiquitin ligase TRAF6 and supports K63-ubiquitination of Akt. The interactome profile of FKBP51 from melanoma cells highlights a relevant role for PHLPP in improving oncogenic hallmarks, particularly, cell proliferation.

Alternative splicing of FKBP5 gene exerts control over T lymphocyte expansion.

FKBP51 is constitutively expressed by immune cells. As other FKBP family members, FKBP51 acts as a coreceptor for the natural products FK506 and rapamycin, which exhibit immunosuppressive effects. However, little is known about the intrinsic role of this large FKBP in the primary function of lymphocytes, that is, the adaptive immune response against foreign antigens, for example, pathogens. This paper aimed to investigate whether FKBP51 expression was modulated during lymphocyte activation. Moreover, as we recently identified a splicing isoform of FKBP51, namely FKBP51s, we also measured this splice protein, along with the canonical one, at different times of a peripheral blood mononuclear cell culture stimulated via T cell receptor. Our results show that the two FKBP51 isoforms were alternatively induced during the proliferative burst. Canonical FKBP51 increased in the time window between 48 and 96 h and its expression levels correlated with cyclin D levels. FKBP51s transiently increased earlier, at 24-36 h to reappearing later, at 120 h, when cyclin D expression returned at resting levels and proliferation ceased. Interestingly, within these two specific timeframes, FKBP51s accumulated in the nucleus. Here FKBP51s colocalized with the Foxp3 transcription factor at 36 h. Regulatory T cell (Treg) counts significantly decreased when FKBP51s was downmodulated. The coculture suppression assay suggested that FKBP51s supports the suppressive capability of Tregs. At 120 h, chromatin immunoprecipitation experiments found FKBP51s linked to CCND1 gene, suggesting a possible effect on gene transcription regulation, as previously demonstrated in melanoma. In conclusion, our study shows that FKBP5 isoforms are upregulated during lymphocyte activation, albeit on different timeframes. The activation of canonical FKBP51 coincides with proliferation hallmarks; FKBP5 splicing occurs early to sustain Treg development and late when proliferation ceases.

NLRP3, the inflammasome and COVID-19 infection.

Severe coronavirus disease 2019 (COVID-19) is characterized by respiratory failure, shock or multiorgan dysfunction, often accompanied by systemic hyperinflammation and dysregulated cytokine release. These features are linked to the intense and rapid stimulation of the innate immune response. The NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome is a central player in inflammatory macrophage activation which via caspase-1 activation leads to the release of the mature forms of the proinflammatory cytokines interleukin (IL)-1ß and IL-18, and via cleavage of Gasdermin D pyroptosis, an inflammatory form of cell death. Here, we discuss the role of NLRP3 activation in COVID-19 and clinical trials currently underway to target NLRP3 to treat severe COVID-19.

Behavioral avoidance of contagious and non-contagious adults.

Evolutionary theories of disease avoidance propose that humans have a set of universal psychological processes to detect environmental cues indicative of infectious disease. These processes then initiate cognitive, emotional, and behavioral responses that function to limit contact with harmful pathogens. Here, we study the conditions under which people exhibit behavioral avoidance of others with a contagious illness or a physical injury (i.e., a broken leg), and the potential mechanisms that underlie this behavior. Across three studies, participants were given the option of sitting at one of two workstations previously occupied by two confederates, one of whom either showed visible symptoms of a cold (contagion condition), wore a lower-leg orthopedic boot and used crutches (broken leg condition), or showed no signs of illness or physical injury (control). We found strong evidence that adults explicitly avoid contact with individuals who show symptoms of a contagious illness. Further, we provide some evidence that adults also avoid individuals with a physical injury, but that this behavior might be driven by implicit, unconscious processes. The findings are discussed in terms of implications for the healthy avoidance of contagion, and the risk for potential stigmatization of non-contagious groups.

Death Related to a Congenital Vascular Anomaly of Pulmonary Hamartoma Type: Malpractice or Tragic Fatality?

In forensic pathology, apparently straightforward cases can often hide rarities that, if not correctly interpreted, can alter the results of the entire investigation, leading to misinterpretations. This occurs when the investigation is conducted to assess medical malpractice. An unexpected death, with no known apparent cause, is often linked to an underlying disease process of unclear etiological origin whose nature can, unfortunately, be properly investigated only post-mortem. This presentation shows a case study, in which it was possible to reconduct the death of a patient to a natural pathology and not to medical treatment. Here, the authors illustrate a case with a hamartoma developed in chronic inflammatory conditions (bronchiectasis) that was difficult to differentiate from lung cancer due to the inability to perform specific instrumental examinations. The hamartoma, usually benign and identifiable by standard instrumental investigations, in this case, led to the patient's death precisely during the execution of a bronchoscopy. However, in the absence of a certain cause of death, public opinion unanimously attributes a patient's disease to medical error. Indeed, a routine practice such as bronchoscopy should not cause death and consequently, the doctor must have made a mistake. Fortunately, the autopsy not only demonstrated the origin of the bleeding but also unveiled the reason for this, as rare congenital lung disease. Fate, one might say.

PD-L1 Expression Fluctuates Concurrently with Cyclin D in Glioblastoma Cells.

Despite Glioblastoma (GBM) frequently expressing programmed cell death ligand-1 (PD-L1), treatment with anti-programmed cell death-1 (PD1) has not yielded brilliant results. Intratumor variability of PD-L1 can impact determination accuracy. A previous study on mouse embryonic fibroblasts (MEFs) reported a role for cyclin-D in control of PD-L1 expression. Because tumor-cell growth within a cancer is highly heterogeneous, we looked at whether PD-L1 and its cochaperone FKBP51s were influenced by cell proliferation, using U251 and SF767 GBM-cell-lines. PD-L1 was measured by Western blot, flow cytometry, confocal-microscopy, quantitative PCR (qPCR), CCND1 by qPCR, FKBP51s by Western blot and confocal-microscopy. Chromatin-Immunoprecipitation assay (xChIp) served to assess the DNA-binding of FKBP51 isoforms. In the course of cell culture, PD-L1 appeared to increase concomitantly to cyclin-D on G1/S transition, to decrease during exponential cell growth progressively. We calculated a correlation between CCND1 and PD-L1 gene expression levels. In the temporal window of PD-L1 and CCND1 peak, FKBP51s localized in ER. When cyclin-D declined, FKBP51s went nuclear. XChIp showed that FKBP51s binds CCND1 gene in a closed-chromatin configuration. Our finding suggests that the dynamism of PD-L1 expression in GBM follows cyclin-D fluctuation and raises the hypothesis that FKBP51s might participate in the events that govern cyclin-D oscillation.

Long-chain polyphosphates impair SARS-CoV-2 infection and replication.

Inorganic polyphosphates (polyPs) are linear polymers composed of repeated phosphate (PO4 3-) units linked together by multiple high-energy phosphoanhydride bonds. In addition to being a source of energy, polyPs have cytoprotective and antiviral activities. Here, we investigated the antiviral activities of long-chain polyPs against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In molecular docking analyses, polyPs interacted with several conserved amino acid residues in angiotensin-converting enzyme 2 (ACE2), the host receptor that facilitates virus entry, and in viral RNA-dependent RNA polymerase (RdRp). ELISA and limited proteolysis assays using nano- LC-MS/MS mapped polyP120 binding to ACE2, and site-directed mutagenesis confirmed interactions between ACE2 and SARS-CoV-2 RdRp and identified the specific amino acid residues involved. PolyP120 enhanced the proteasomal degradation of both ACE2 and RdRp, thus impairing replication of the British B.1.1.7 SARS-CoV-2 variant. We thus tested polyPs for functional interactions with the virus in SARS-CoV-2-infected Vero E6 and Caco2 cells and in primary human nasal epithelial cells. Delivery of a nebulized form of polyP120 reduced the amounts of viral positive-sense genomic and subgenomic RNAs, of RNA transcripts encoding proinflammatory cytokines, and of viral structural proteins, thereby presenting SARS-CoV-2 infection in cells in vitro.

Prune-1 drives polarization of tumor-associated macrophages (TAMs) within the lung metastatic niche in triple-negative breast cancer.

M2-tumor-associated macrophages (M2-TAMs) in the tumor microenvironment represent a prognostic indicator for poor outcome in triple-negative breast cancer (TNBC). Here we show that Prune-1 overexpression in human TNBC patients has positive correlation to lung metastasis and infiltrating M2-TAMs. Thus, we demonstrate that Prune-1 promotes lung metastasis in a genetically engineered mouse model of metastatic TNBC augmenting M2-polarization of TAMs within the tumor microenvironment. Thus, this occurs through TGF-ß enhancement, IL-17F secretion, and extracellular vesicle protein content modulation. We also find murine inactivating gene variants in human TNBC patient cohorts that are involved in activation of the innate immune response, cell adhesion, apoptotic pathways, and DNA repair. Altogether, we indicate that the overexpression of Prune-1, IL-10, COL4A1, ILR1, and PDGFB, together with inactivating mutations of PDE9A, CD244, Sirpb1b, SV140, Iqca1, and PIP5K1B genes, might represent a route of metastatic lung dissemination that need future prognostic validations.

Early interventions in a US military FIRST episode psychosis program.

AIM: Naval Medical Center San Diego's Psychiatric Transition Program is a specialized first episode psychosis treatment program that delivers coordinated specialty care to military service members with psychotic disorders. Due to the unique military environment, military service members with first episode psychosis are hypothesized to receive care very early after the emergence of first psychotic symptoms, resulting in significantly reduced duration of untreated psychosis. This study's aim is to calculate the duration of untreated psychosis for patients enrolled in Naval Medical Center San Diego's Psychiatric Transition Program (NMCSD PTP) from 01JUL2014-31DEC2016. METHODS: Patients included in this study had a diagnosis of schizophreniform disorder (13.04%), schizophrenia (43.48%), schizoaffective disorder (8.70%), other specified schizophreniform disorder (30.43%), or brief psychotic disorder (4.35%) upon discharge from military service and NMCSD PTP. Duration of untreated psychosis was defined as the interval from emergence of positive psychotic symptoms to antipsychotic medication initiation. Duration of untreated psychosis was measured through retrospective review of the electronic medical record. A total of 69 subjects in the Naval Medical Center San Diego's Psychiatric Transition Program met inclusion criteria. Mean and median values as well as standard deviations were calculated for all included subjects. RESULTS: The mean duration to scheduled (non-PRN) neuroleptic medication was 37 days (median: 4 days). The mean duration to PRN neuroleptic medication was 21 days (median: 2 days). CONCLUSIONS: These data support our view that the structure of the military and military healthcare system markedly shortens the DUP for military service members who experience first episode psychosis.

Structural and Kinetic Studies of the Potent Inhibition of Metallo-ß-lactamases by 6-Phosphonomethylpyridine-2-carboxylates.

There are currently no clinically available inhibitors of metallo-ß-lactamases (MBLs), enzymes that hydrolyze ß-lactam antibiotics and confer resistance to Gram-negative bacteria. Here we present 6-phosphonomethylpyridine-2-carboxylates (PMPCs) as potent inhibitors of subclass B1 (IMP-1, VIM-2, and NDM-1) and B3 (L1) MBLs. Inhibition followed a competitive, slow-binding model without an isomerization step (IC50 values of 0.3-7.2 µM; Ki values of 0.03-1.5 µM). Minimum inhibitory concentration assays demonstrated potentiation of ß-lactam (Meropenem) activity against MBL-producing bacteria, including clinical isolates, at concentrations at which eukaryotic cells remain viable. Crystal structures revealed unprecedented modes of binding of inhibitor to B1 (IMP-1) and B3 (L1) MBLs. In IMP-1, binding does not replace the nucleophilic hydroxide, and the PMPC carboxylate and pyridine nitrogen interact closely (2.3 and 2.7 Å, respectively) with the Zn2 ion of the binuclear metal site. The phosphonate group makes limited interactions but is 2.6 Å from the nucleophilic hydroxide. Furthermore, the presence of a water molecule interacting with the PMPC phosphonate and pyridine N-C2 π-bond, as well as the nucleophilic hydroxide, suggests that the PMPC binds to the MBL active site as its hydrate. Binding is markedly different in L1, with the phosphonate displacing both Zn2, forming a monozinc enzyme, and the nucleophilic hydroxide, while also making multiple interactions with the protein main chain and Zn1. The carboxylate and pyridine nitrogen interact with Ser221 and -223, respectively (3 Å distance). The potency, low toxicity, cellular activity, and amenability to further modification of PMPCs indicate these and similar phosphonate compounds can be further considered for future MBL inhibitor development.

Arginine-containing peptides as potent inhibitors of VIM-2 metallo-ß-lactamase.

BACKGROUND: Metallo-ß-lactamases (MBLs) play an important role in the emergence of microbial resistance to ß-lactam antibiotics, and are hence considered targets for the design of novel therapeutics. We here report on the inhibitory effect of peptides containing multiple arginine residues on VIM-2, a clinically important MBL from Pseudomonas aeruginosa. METHODS: Enzyme kinetic assays in combination with fluorescence spectroscopy and stopped-flow UV-Vis spectrophotometry were utilized to explore the structure-activity relationship of peptides as inhibitors of VIM-2. RESULTS: Our studies show that the inhibitory potency of the investigated peptides was mainly dependent on the number of arginine residues in the center of the peptide sequence, and on the composition of the N-terminus. The most potent inhibitors were found to curtail enzyme function in the mid-to-low nanomolar range. Salts generally reduced peptide-mediated inhibition. Analysis of the mode of inhibition suggests the peptides to act as mixed-type inhibitors with a higher affinity for the enzyme-substrate complex. Stopped-flow UV-Vis and fluorescence studies revealed the peptides to induce rapid protein aggregation, a phenomenon strongly correlated to the peptides' inhibitory potency. Inhibition of IMP-1 (another subclass B1 MBL) by the peptides was found to be much weaker than that observed with VIM-2, a finding which might be related to subtle molecular differences in the protein surfaces. CONCLUSION: The reported data indicate that arginine-containing peptides can serve as potent, aggregation-inducing inhibitors of VIM-2, and potentially of other MBLs. GENERAL SIGNIFICANCE: Arginine-containing peptides can be considered as a novel type of potent MBL inhibitors.

Assay for drug discovery: Synthesis and testing of nitrocefin analogues for use as ß-lactamase substrates.

We report on the synthesis of three nitrocefin analogues and their evaluation as substrates for the detection of ß-lactamase activity. These compounds are hydrolyzed by all four Ambler classes of ß-lactamases. Kinetic parameters were determined with eight different ß-lactamases, including VIM-2, NDM-1, KPC-2, and SPM-1. The compounds do not inhibit the growth of clinically important antibiotic-resistant gram-negative bacteria in vitro. These chromogenic compounds have a distinct absorbance spectrum and turn purple when hydrolyzed by ß-lactamases. One of these compounds, UW154, is easier to synthesize from commercial starting materials than nitrocefin and should be significantly less expensive to produce.

Clinical outcomes of diabetic patients at a student-run free clinic project.

OBJECTIVES: Our objective was to determine if the quality of care of diabetic patients at a Student-Run Free Clinic Project (SRFCP) meets the standard of care, is comparable with other published outcomes, and whether pertinent diabetic clinical indicators improve over time. METHODS: The authors conducted a retrospective chart review of diabetic patients at three University of California-San Diego (UCSD) SRFCP sites from December 1, 2008 to December 1, 2009 (n=182), calculated the percentage who received recommended screening tests, percent at goal, and compared these to published outcomes using Fisher's exact tests. Baseline measures were compared to most recent values using paired t tests. RESULTS: The percentage of patients who received recommended screening tests (process measures) was blood pressure (BP) 100%, HbA1c 99.5%, creatinine 99.5%, LDL 93%, HDL and triglycerides 88%, microalbumin/creatinine ratio 80%, and ophthalmology exam 32%. Intermediate outcomes included: 70% of patients were at LDL goal <100, 70% had microalbumin/creatinine ratio <30, 61% of males were at HDL goal %gt;40, 47% of females at HDL goal>50, 52% with triglycerides <150, 45% had BP <130/80, and 30% of patients had HbA1c <7. Mean HbA1c, LDL, HDL, triglycerides and blood pressure improved significantly over time. CONCLUSIONS: Diabetic patients at UCSD SRFCP reached goals for both process measures and intermediate outcomes at rates that meet or exceed published outcomes of insured and uninsured diabetics on nearly all measures, with the exception of ophthalmology screening. Glycemic control, cholesterol levels, and blood pressure improved significantly during care at the UCSD SRFCP.

Cyclobutanone analogues of beta-lactams revisited: insights into conformational requirements for inhibition of serine- and metallo-beta-lactamases.

The most important mode of bacterial resistance to beta-lactam antibiotics is the expression of beta-lactamases. New cyclobutanone analogues of penams and penems have been prepared and evaluated for inhibition of class A, B, C, and D beta-lactamases. Inhibitors which favor conformations in which the C4 carboxylate is equatorial were found to be more potent than those in which the carboxylate is axial, and molecular modeling studies with enzyme-inhibitor complexes indicate that an equatorial orientation of the carboxylate is required for binding to beta-lactamases. An X-ray structure of OXA-10 complexed with a cyclobutanone confirms that a serine hemiketal is formed in the active site and that the inhibitor adopts the exo envelope. An unsaturated penem analogue was also found to enhance the potency of meropenem against carbapenem-resistant MBL-producing strains of Chryseobacterium meningosepticum and Stenotrophomonas maltophilia. These cyclobutanones represent the first type of reversible inhibitors to show moderate (low micromolar) inhibition of both serine- and metallo-beta-lactamases and should be considered for further development into practical inhibitors.

Assays for beta-lactamase activity and inhibition.

The ability, either innate or acquired, to produce beta-lactamases, enzymes capable of hydrolyzing the endocyclic peptide bond in beta-lactam antibiotics, would appear to be a primary contributor to the ever-increasing incidences of resistance to this class of antibiotics. To date, four distinct classes, A, B, C, and D, of beta-lactamases have been identified. Of these, enzymes in classes A, C, and D utilize a serine residue as a nucleophile in their catalytic mechanism while class B members are Zn2+-dependent for their function. Efforts have been and still continue to be made toward the development of potent inhibitors of these enzymes as a means to ensure the efficacy of beta-lactam antibiotics in clinical medicine. This chapter concerns procedures for the evaluation of the catalytic activity of beta-lactamases as a means to screen compounds for their inhibitory potency.

N-arylsulfonyl hydrazones as inhibitors of IMP-1 metallo-beta-lactamase.

Members of a family of N-arylsulfonyl hydrazones have been identified as novel inhibitors of IMP-1, a metallo-beta-lactamase of increasing prevalence. Structure-activity relationship studies have indicated a requirement for bulky aromatic substituents on each side of the sulfonyl hydrazone backbone for these compounds to serve as efficient inhibitors of IMP-1. Molecular modeling has provided insight into the structural basis for the anti-metallo-beta-lactamase activity exhibited by this class of compounds.