Inhibitor-Resistant Mutants Give Important Insights into Candida albicans ABC Transporter Cdr1 Substrate Specificity and Help Elucidate Efflux Pump Inhibition.

Overexpression of ATP-binding cassette (ABC) transporters is a major cause of drug resistance in fungal pathogens. Milbemycins, enniatin B, beauvericin, and FK506 are promising leads for broad-spectrum fungal multidrug efflux pump inhibitors. The characterization of naturally generated inhibitor-resistant mutants is a powerful tool to elucidate structure-activity relationships in ABC transporters. We isolated 20 Saccharomyces cerevisiae mutants overexpressing Candida albicans ABC pump Cdr1 variants resistant to fluconazole efflux inhibition by milbemycin α25 (8 mutants), enniatin B (8), or beauvericin (4). The 20 mutations were in just 9 residues at the centers of transmembrane segment 1 (TMS1) (6 mutations), TMS4 (4), TMS5 (4), TMS8 (1), and TMS11 (2) and in A713P (3), a previously reported FK506-resistant "hot spot 1" mutation in extracellular loop 3. Six Cdr1-G521S/C/V/R (TMS1) variants were resistant to all four inhibitors, four Cdr1-M639I (TMS4) variants were resistant to milbemycin α25 and enniatin B, and two Cdr1-V668I/D (TMS5) variants were resistant to enniatin B and beauvericin. The eight milbemycin α25-resistant mutants were altered in four amino acids as follows: G521R, M639I, A713P, and T1355N (TMS11). These four Cdr1 variants responded differently to various types of inhibitors, and each exhibited altered substrate specificity and kinetic properties. The data infer an entry gate function for Cdr1-G521 and a role for Cdr1-A713 in the constitutively high Cdr1 ATPase activity. Cdr1-M639I and -T1355N possibly cause inhibitor resistance by altering TMS contacts near the substrate/inhibitor-binding pocket. Models for the interactions of substrates and different types of inhibitors with Cdr1 at various stages of the transport cycle are presented.

Identification and functional characterization of Penicillium marneffei major facilitator superfamily (MFS) transporters.

PENICILLIUM MARNEFFEI: is a thermally dimorphic fungus that causes penicilliosis, and become the third-most-common opportunistic fungal infection in immunocompromised patients in Southeast Asia. Azoles and amphotericin B have been introduced for the treatment, however, it is important to investigate possible mechanisms of azole resistance for future treatment failure. We identified 177 putative MFS transporters and classified into 17 subfamilies. Among those, members of the Drug:H+ antiporter 1 subfamily are known to confer resistance to antifungals. Out of 39 paralogs, three (encoded by PmMDR1, PmMDR2, and PmMDR3) were heterologously overexpressed in S. cerevisiae AD∆ conferred resistance to various drugs and compounds including azoles, albeit to different degrees. PmMDR1-expressing strain showed resistance to the broadest range of drugs, followed by the PmMDR3, and PmMDR2 conferred weak resistance to a limited range of drugs. We conclude that PmMDR1 and PmMDR3, may be able to serve as multidrug efflux pumps.

FK506 Resistance of Saccharomyces cerevisiae Pdr5 and Candida albicans Cdr1 Involves Mutations in the Transmembrane Domains and Extracellular Loops.

The 23-membered-ring macrolide tacrolimus, a commonly used immunosuppressant, also known as FK506, is a broad-spectrum inhibitor and an efflux pump substrate of pleiotropic drug resistance (PDR) ATP-binding cassette (ABC) transporters. Little, however, is known about the molecular mechanism by which FK506 inhibits PDR transporter drug efflux. Thus, to obtain further insights we searched for FK506-resistant mutants of Saccharomyces cerevisiae cells overexpressing either the endogenous multidrug efflux pump Pdr5 or its Candida albicans orthologue, Cdr1. A simple but powerful screen gave 69 FK506-resistant mutants with, between them, 72 mutations in either Pdr5 or Cdr1. Twenty mutations were in just three Pdr5/Cdr1 equivalent amino acid positions, T550/T540 and T552/S542 of extracellular loop 1 (EL1) and A723/A713 of EL3. Sixty of the 72 mutations were either in the ELs or the extracellular halves of individual transmembrane spans (TMSs), while 11 mutations were found near the center of individual TMSs, mostly in predicted TMS-TMS contact points, and only two mutations were in the cytosolic nucleotide-binding domains of Pdr5. We propose that FK506 inhibits Pdr5 and Cdr1 drug efflux by slowing transporter opening and/or substrate release, and that FK506 resistance of Pdr5/Cdr1 drug efflux is achieved by modifying critical intramolecular contact points that, when mutated, enable the cotransport of FK506 with other pump substrates. This may also explain why the 35 Cdr1 mutations that caused FK506 insensitivity of fluconazole efflux differed from the 13 Cdr1 mutations that caused FK506 insensitivity of cycloheximide efflux.

Analysis of key clinical features for achieving complete remission in stage III and IV non-small cell lung cancer patients.

BACKGROUND: Although development of immune checkpoint inhibitors and various molecular target agents has extended overall survival time (OS) in advanced non-small cell lung cancer (NSCLC), a complete cure remains rare. We aimed to identify features and treatment modalities of complete remission (CR) cases in stages III and IV NSCLC by analyzing long-term survivors whose OS exceeded 3 years. METHODS: From our hospital database, 1,699 patients, registered as lung cancer between 1st Mar 2004 and 30th Apr 2011, were retrospectively examined. Stage III or IV histologically or cytologically confirmed NSCLC patients with chemotherapy initiated during this period were enrolled. A Cox proportion hazards regression model was used. Data collection was closed on 13th Feb 2017. RESULTS: There were 164 stage III and 279 stage IV patients, including 37 (22.6%) and 51 (18.3%) long-term survivors and 12 (7.3%) and 5 (1.8%) CR patients, respectively. The long-term survivors were divided into three groups: 3 ≤ OS < 5 years, 5 years ≤ OS with tumor, and 5 years ≤ OS without tumor (CR). The median OS of these groups were 1,405, 2,238, and 2,876 days in stage III and 1,368, 2,503, and 2,643 days in stage IV, respectively. The mean chemotherapy cycle numbers were 16, 20, and 10 in stage III and 24, 25, and 5 in stage IV, respectively. In the stage III CR group, all patients received chemoradiation, all oligometastases were controlled by radiation, and none had brain metastases. Compared with non-CR patients, the stage IV CR patients had smaller primary tumors and fewer metastases, which were independent prognostic factors for OS among long-term survivors. The 80% stage IV CR patients received radiation or surgery for controlling primary tumors, and the surgery rate for oligometastases was high. Pathological findings in the stage IV CR patients revealed that numerous inflammatory cells existed around and inside resected lung and brain tumors, indicating strong immune response. CONCLUSIONS: Multiple line chemotherapies with primary and oligometastatic controls by surgery and/or radiation might achieve cure in certain advanced NSCLC. Cure strategies must be changed according to stage III or IV. This study was retrospectively registered on 16th Jun 2019 in UMIN Clinical Trials Registry (number UMIN000037078).

Identification and characterization of Candida utilis multidrug efflux transporter CuCdr1p.

The edible, nitrate assimilating, yeast Candida utilis is a commercial food additive, and it is a potentially useful host for heterologous protein expression. A number of ATP-binding cassette (ABC) transporters are multidrug efflux pumps that can cause multidrug resistance in opportunistic pathogens. In order to develop optimal novel antimicrobial agents it is imperative to understand the structure, function and expression of these transporters. With the ultimate aim of developing an alternative yeast host for the heterologous expression of eukaryotic membrane transporters, and to identify ABC transporters potentially associated with C. utilis multidrug resistance, we classified the entire repertoire of 30 C. utilis ABC proteins. We named the open reading frame most similar to the archetype multidrug efflux pump gene C. albicans CDR1 as CuCDR1 Overexpression of CuCDR1 in Saccharomyces cerevisiae ADΔ caused multidrug resistance similar to that of cells overexpressing CaCDR1 Unlike CaCdr1p, however, the C-terminally green fluorescent protein (GFP) tagged CuCdr1p-GFP was functionally impaired and did not properly localize to the plasma membrane. CuCdr1p function could be recovered however by adding a 15 amino acid linker -GAGGSAGGSGGAGAG- between CuCdr1p and the C-terminal GFP tag.

Identification and functional characterization of Penicillium marneffei pleiotropic drug resistance transporters ABC1 and ABC2.

Penicilliosis caused by the dimorphic fungus Penicillium marneffei is an endemic, AIDS-defining illness and, after tuberculosis and cryptococcosis, the third most common opportunistic infection of AIDS patients in tropical Southeast Asia. Untreated, patients have poor prognosis; however, primary amphotericin B treatment followed by prolonged itraconazole prophylaxis is effective. To identify ATP-binding cassette (ABC) transporters that may play a role in potential multidrug resistance of P. marneffei, we identified and classified all 46 P. marneffei ABC transporters from the genome sequence. PmABC1 and PmABC2 were most similar to the archetype Candida albicans multidrug efflux pump gene CDR1. P. marneffei Abc1p (PmAbc1p) was functionally expressed in Saccharomyces cerevisiae, although at rather low levels, and correctly localized to the plasma membrane, causing cells to be fourfold to eightfold more resistant to azoles and many other xenobiotics than untransformed cells. P. marneffei Abc2p (PmAbc2p) was expressed at similarly low levels, but it had no efflux activity and did not properly localize to the plasma membrane. Interestingly, PmAbc1p mislocalized and lost its transport activity when cells were shifted to 37 °C. We conclude that expression of PmAbc1p in S. cerevisiae confers resistance to several xenobiotics indicating that PmAbc1p may be a multidrug efflux pump.

Dysregulated synthesis of protectin D1 in eosinophils from patients with severe asthma.

BACKGROUND: Protectin D1 (PD1) is an anti-inflammatory and proresolving lipid mediator biosynthesized from the omega-3 fatty acid docosahexaenoic acid (DHA). Exogenous PD1 conferred protection against eosinophilic inflammation in animals with experimental asthma, although its endogenous cellular source and functions in human airways are of interest. OBJECTIVE: We sought to investigate the synthesizing capacity of PD1 in eosinophils from healthy subjects and patients with severe asthma and its direct effects on eosinophil functions. METHODS: Human eosinophil-derived metabolites of arachidonic acid and DHA were analyzed with liquid chromatography-tandem mass spectrometry-based lipidomic analysis. The biological activities of PD1 on the function of human eosinophils, including chemotaxis, adhesion molecule expressions, degranulation, superoxide anion generation, or survival, were examined. RESULTS: We identified PD1 as one of the main anti-inflammatory and proresolving molecules synthesized in human eosinophils. PD1, in nanomolar concentrations, suppressed the chemotaxis induced by CCL11/eotaxin-1 or 5-oxo-eicosatetraenoic acid and modulated the expression of the adhesion molecules CD11b and L-selectin, although it had no effects on the degranulation, superoxide anion generation, or survival of the eosinophils. Compared with the cells harvested from healthy subjects, we observed a prominent decrease in the biosynthesis of PD1 by eosinophils from patients with severe asthma, even in presence of DHA. CONCLUSION: These observations are a first indication that activated human eosinophils represent a major source of PD1, which can act as a self-resolving machinery in eosinophilic inflammation, whereas the production of PD1 is impaired in patients with severe asthma.

Specific interactions between the Candida albicans ABC transporter Cdr1p ectodomain and a D-octapeptide derivative inhibitor.

Overexpression of the Candida albicans ATP-binding cassette transporter CaCdr1p causes clinically significant resistance to azole drugs including fluconazole (FLC). Screening of a ~1.89 × 10(6) member D-octapeptide combinatorial library that concentrates library members at the yeast cell surface identified RC21v3, a 4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative of the D-octapeptide D-NH(2) -FFKWQRRR-CONH(2) , as a potent and stereospecific inhibitor of CaCdr1p. RC21v3 chemosensitized Saccharomyces cerevisiae strains overexpressing CaCdr1p but not other fungal ABC transporters, the C. albicans MFS transporter CaMdr1p or the azole target enzyme CaErg11p, to FLC. RC21v3 also chemosensitized clinical C. albicans isolates overexpressing CaCDR1 to FLC, even when CaCDR2 was overexpressed. Specific targeting of CaCdr1p by RC21v3 was confirmed by spontaneous RC21v3 chemosensitization-resistant suppressor mutants of S. cerevisiae expressing CaCdr1p. The suppressor mutations introduced a positive charge beside, or within, extracellular loops 1, 3, 4 and 6 of CaCdr1p or an aromatic residue near the extracytoplasmic end of transmembrane segment 5. The mutations did not affect CaCdr1p localization or CaCdr1p ATPase activity but some increased susceptibility to the CaCdr1p substrates FLC, rhodamine 6G, rhodamine 123 and cycloheximide. The suppressor mutations showed that the drug-like CaCdr1p inhibitors FK506, enniatin, milbemycin α11 and milbemycin ß9 have modes of action similar to RC21v3.

Adhesion of Yeast and Bacteria to Oral Surfaces.

Colonization of surfaces in the human body by microorganisms is an early, essential, step in the initiation of infectious disease. We have developed in vitro assays to investigate interactions between yeast or bacterial cells and human tissues, fluids, or prostheses. Such assays can be used to identify the adhesins, ligands, and receptors involved in these interactions, for example, by determining which components of the microbe or human tissue/fluid interfere with adherence in the assay. The assays can also be applied to find ways of preventing adhesion, and subsequent disease, by investigating the effects of different conditions and added compounds on adherence in the in vitro assays. Here we describe assays for measuring adhesion of the oral yeast Candida albicans, a common commensal and opportunistic pathogen, or the bacterium Staphylococcus epidermidis, which is not normally pathogenic but is known to form biofilms on medical prostheses. The assays described belong to two approaches to investigating adhesion and biofilm formation: (i) retention at a fixed time point following liquid washes, and (ii) retention against a continuous flow of medium.

Retroperitoneal Hematoma During Prophylactic Dose of Heparin Therapy for Coronavirus Disease 2019.

We report a case of retroperitoneal hematoma during prophylactic heparin therapy for coronavirus disease 2019 (COVID-19). A 79-year-old man was diagnosed with COVID-19 pneumonia with possible exacerbation of fibrotic hypersensitivity pneumonia. He received a prophylactic dose of subcutaneous heparin therapy, methylprednisolone pulse therapy and Intravenous remdesivir but developed a spontaneous iliopsoas muscle hematoma, and transcatheter arterial embolization was performed. Even with a prophylactic dose of subcutaneous heparin therapy, the course should be carefully monitored, especially in patients with preexisting risk factors for hemorrhagic complications. Once retroperitoneal hematoma develops, aggressive procedures, such as transcatheter arterial embolization, should be considered to avoid fatal outcomes.

Nodular Pulmonary Amyloidosis Preceding Gastric Mucosa-associated Lymphoid Tissue Lymphoma, Initially Suspected to Be Lung Cancer.

Nodular pulmonary amyloidosis, a subtype of pulmonary amyloidosis, is a unique disease that can mimic lung cancer on radiographic imaging and is related to lymphoproliferative disorders. In this report, we describe a case of a 76-year-old male who presented with a solitary nodule in his left lower lung lobe on computed tomography that increased from 6 mm to 13 mm in diameter over 40 months. Lung cancer was suspected; however, transbronchial lung biopsy revealed deposition of an eosinophilic and homogeneous amorphous substance, which showed apple-green birefringence under polarized light after Congo red staining, and immunohistochemistry analysis returned positive results for immunoglobulin lambda light-chain. Upper gastrointestinal endoscopy revealed a gastric mucosa-associated lymphoid tissue (MALT) lymphoma. These findings indicated that this was a case of nodular pulmonary amyloidosis that preceded a diagnosis of MALT lymphoma.

The phosphoinositide 3'-kinase p110δ modulates contractile protein production and IL-6 release in human airway smooth muscle.

Transforming growth factor (TGF) ß1 increases pro-inflammatory cytokines and contractile protein expression by human airway smooth muscle (ASM) cells, which could augment airway inflammation and hyperresponsiveness. Phosphoinositide 3' kinase (PI3K) is one of the signaling pathways implicated in TGFß1 stimulation, and may be altered in asthmatic airways. This study compared the expression of PI3K isoforms by ASM cells from donors with asthma (A), chronic obstructive pulmonary disease (COPD), or neither disease (NA), and investigated the role of PI3K isoforms in the production of TGFß1 induced pro-inflammatory cytokine and contractile proteins in ASM cells. A cells expressed higher basal levels of p110δ mRNA compared to NA and COPD cells; however COPD cells produced more p110δ protein. TGFß1 increased 110δ mRNA expression to the same extent in the three groups. Neither the p110δ inhibitor IC87114 (1, 10, 30 µM), the p110ß inhibitor TGX221 (0.1, 1, 10 µM) nor the PI3K pan inhibitor LY294002 (3, 10 µM) had any effect on basal IL-6, calponin or smooth muscle α-actin (α-SMA) expression. However, TGFß1 increased calponin and α-SMA expression was inhibited by IC87114 and LY294002 in all three groups. IC87114, TGX221, and LY294002 reduced TGFß1 induced IL-6 release in a dose related manner in all groups of ASM cells. PI3K p110δ is important for TGFß1 induced production of the contractile proteins calponin and α-SMA and the proinflammatory cytokine IL-6 in ASM cells, and may therefore be relevant as a potential therapeutic target to treat both inflammation and airway remodeling.

ß2-Agonists upregulate PDE4 mRNA but not protein or activity in human airway smooth muscle cells from asthmatic and nonasthmatic volunteers.

ß(2)-Adrenergic receptor (ß2AR) agonists induce airway relaxation via cAMP. Phosphodiesterase (PDE)s degrade and regulate cAMP, and in airway smooth muscle (ASM) cells PDE4D degrades cAMP. Long-acting ß(2)-agonists are now contraindicated as monotherapy for asthma, and increased PDE4D has been speculated to contribute to this phenomenon. In this study we investigated the expression of PDE4D in asthmatic and nonasthmatic ASM cells and its regulation by formoterol and budesonide. Primary ASM cells from people with or without asthma were stimulated with transforming growth factor (TGF)-ß(1), formoterol, and/or budesonide. PDE4D mRNA was assessed by real-time PCR, or PCR to assess splice variant production. PDE4D protein was assessed by Western blotting, and we investigated the effect of formoterol on cAMP production and PDE activity. Interleukin (IL)-6 was assessed using ELISA. PDE4D mRNA was dose dependently upregulated by formoterol, with a single splice variant, PDE4D5, present. Formoterol did not induce PDE4D protein at time points between 3 to 72 h, whereas it did induce and increase IL-6 secretion. We pretreated cells with actinomycin D and a proteasome inhibitor, MG132, and found no evidence of alterations in mRNA, protein expression, or degradation of PDE4D. Finally PDE activity was not altered by formoterol. This study shows, for the first time, that PDE4D5 is predominantly expressed in human ASM cells from people with and without asthma and that formoterol does not upregulate PDE4D protein production. This leads us to speculate that continual therapy with ß2AR agonists is unlikely to cause PDE4-mediated tachyphylaxis.

Reconstitution of high-level micafungin resistance detected in a clinical isolate of Candida glabrata identifies functional homozygosity in glucan synthase gene expression.

OBJECTIVES: A mechanism for the acquisition of high-level echinocandin resistance in Candida glabrata was investigated. FKS mutants were constructed to: determine whether clinically significant micafungin resistance requires a hot-spot mutation in FKS1 and a premature stop codon in FKS2, as was observed in a clinical isolate; select for variants with reduced susceptibility and locate mutations in FKS genes; and assess the roles of FKS1 and FKS2. METHODS: A panel of FKS mutants was constructed using micafungin-susceptible parents by site-directed mutagenesis. Drug susceptibility, gene expression and glucan synthase activities were compared between mutants. Mutations acquired by selection were identified by DNA sequence analysis of FKS genes from selected variants. Single FKS deletants were constructed and their phenotypes examined. RESULTS: Introduction of the hot-spot mutation in FKS1 alone conferred an intermediate reduction in susceptibility, and the premature stop codon in FKS2 alone had no effect on susceptibility, while severely reduced susceptibility equivalent to that of the clinical isolate required both mutations. Exposure of susceptible strains to micafungin yielded variants with an intermediate reduction in susceptibility that possessed a hot-spot mutation in FKS1. Further exposure to micafungin yielded variants with severely reduced susceptibility that acquired various single mutations in FKS2. The phenotypes of Δfks1 and Δfks2 mutants indicate that the two FKS genes are functionally redundant, while deletion of both FKS1 and FKS2 conferred synthetic lethality. CONCLUSIONS: In the laboratory mutants of C. glabrata, clinically significant reduced susceptibility to micafungin required single nucleotide changes in both FKS1 and FKS2, and both genes encoded ß-1,3-glucan synthase catalytic subunits.

Palliative Care Intervention for End-of-life Patients with Non-cancerous Respiratory Diseases.

While palliative care for patients with cancer is actively performed, it is provided only occasionally for patients with chronic non-cancerous respiratory diseases. This is due to various factors, including the fact that palliative care is not covered by health insurance and the difficulty in determining end-of-life in these patients. This paper presents two case studies to highlight the significance of palliative care team intervention for patients in the terminal stage of chronic non-cancerous respiratory diseases. Palliative care is essential to support physical problems, such as dyspnea, as well as mental disorders, such as depression, and to provide nutrition therapy and rehabilitation. To achieve care at the appropriate time in accordance with the patient's wishes, it is essential for patients to understand and accept the progress and deterioration of their disease and prepare for the end of life at an earlier stage under multidisciplinary involvement (advance care planning).

Rhabdomyolysis Caused by Gefitinib Overdose.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are common therapeutic agents for EGFR mutation-positive advanced non-small-cell lung cancer. There has been no report of rhabdomyolysis caused by an overdose of EGFR-TKIs. We herein review the existing literature on the subject and report a rare case of rhabdomyolysis due to an overdose of gefitinib, an EGFR-TKI.

Efficacy of Prophylactic Antibiotic Use in Preventing Post-bronchoscopy Pneumonia in Lung Cancer Patients.

OBJECTIVE: Post-bronchoscopy pneumonia can affect the prognosis of lung cancer patients. This prospective study examined the efficacy of prophylactic antibiotics for lung cancer patients at high-risk of post-bronchoscopy pneumonia, determined by our prediction score, using three risk factors: age 70 years or older, current smoking, and central tumors visualized on CT. METHODS: Patients with lung cancer who underwent diagnostic bronchoscopy between June 2018 and March 2020 with a score of 2 points or higher were enrolled. Sulbactam/ampicillin was administered intravenously within one hour prior to bronchoscopy, followed by oral clavulanate/amoxicillin for three days. We used the data of lung cancer patients who underwent diagnostic bronchoscopy between April 2012 and July 2014 and exhibited a score of 2 or higher as the historical control. RESULTS: Post-bronchoscopy pneumonia occurred in none of the 24 patients in the prophylaxis group and in 17 of 144 patients in the control group, with no significant difference in the incidence of pneumonia between the two groups. CONCLUSIONS: Antibiotic prophylaxis can be effective and safe for the patients high-risk of post-bronchoscopy pneumonia. A multicenter prospective study to examine the effects of prophylactic antibiotics in high-risk patients is feasible with a modest number of participants.

ANCA-Associated Vasculitis in a Patient with Chronic Thromboembolic Pulmonary Hypertension.

An 82-year-old woman with a history of chronic thromboembolic pulmonary hypertension (CTEPH) presented with malaise, left facial nerve paralysis and the positive seroconversion of myeloperoxidase (MPO)-antineutrophil cytoplasmic antibody (ANCA). She was diagnosed with ANCA-associated vasculitis (AAV). Administration of corticosteroids significantly improved her symptoms, with a decline in the serum MPOANCA level. Ten months later than the initial presentation, she developed an AAV exacerbation with lung infiltration and pericardial effusion, which improved with high-dose corticosteroid therapy. To date, a limited number of AAV cases concomitant with pulmonary hypertension have been reported. The case report presented herein suggests a potential role for CTEPH in the development of AAV.

Cryptogenic Fibrosing Pleuritis with Rapidly Progressive Restrictive Ventilatory Dysfunction.

Cryptogenic bilateral fibrosing pleuritis is a rare condition, and its pathogenesis and clinical course are poorly understood, with no established therapy available. A 61-year-old man presented with bilateral pleural thickening and lymphocytic exudative effusions. The patient was diagnosed with fibrosing pleuritis with no evidence of a known etiology on a surgical pleural biopsy. Within 16 months from the onset of respiratory symptoms, restrictive ventilatory impairment progressed rapidly, resulting in hypercapnic respiratory failure requiring home oxygen and non-invasive positive pressure ventilation therapies.