Candida albicans Promotes the Antimicrobial Tolerance of Escherichia coli in a Cross-Kingdom Dual-Species Biofilm

Cross-kingdom multi-species biofilms consisting of fungi and bacteria are often resistant to antimicrobial treatment, leading to persistent infections. We evaluated whether the presence of Candida albicans affects the antibacterial tolerance of Escherichia coli in dual-species biofilms and explored the underlying mechanism. We found that the survival of E. coli in the presence of antibacterial drugs was higher in dual-species biofilms compared to single-species biofilms. This tolerance-inducing effect was observed in E. coli biofilms that were treated with a C. albicans culture supernatant. To explore the antibacterial tolerance-inducing factor contained in the culture supernatant and identify the tolerance mechanism, a heated supernatant, a supernatant treated with lyticase, DNase, and proteinase K, or a supernatant added to a drug efflux pump inhibitor were used. However, the tolerance-inducing activity was not lost, indicating the existence of some other mechanisms. Ultrafiltration revealed that the material responsible for tolerance-inducing activity was <10 kDa in size. This factor has not yet been identified and needs further studies to understand the mechanisms of action of this small molecule precisely. Nevertheless, we provide experimental evidence that Candida culture supernatant induces E. coli antibacterial tolerance in biofilms. These findings will guide the development of new treatments for dual-species biofilm infections.

Characteristics of aspiration pneumonia patients in acute care hospitals: A multicenter, retrospective survey in Northern Japan.

BACKGROUND: Pneumonia is a common cause of illness and death of the elderly in Japan. Its prevalence is escalating globally with the aging of population. To describe the latest trends in pneumonia hospitalizations, especially aspiration pneumonia (AP) cases, we assessed the clinical records of pneumonia patients admitted to core acute care hospitals in Miyagi prefecture, Japan. METHODS: A retrospective multi-institutional joint research was conducted for hospitalized pneumonia patients aged ≥20 years from January 2019 to December 2019. Clinical data of patients were collected from the medical records of eight acute care hospitals. RESULTS: Out of the 1,800 patients included in this study, 79% of the hospitalized pneumonia patients were aged above 70 years. The most common age group was in the 80s. The ratio of AP to total pneumonia cases increased with age, and 692 out of 1,800 patients had AP. In univariate analysis, these patients had significantly older ages, lower body mass index (BMI), a lower ratio of normal diet intake and homestay before hospitalization, along with more AP recurrences and comorbidities. During hospitalization, AP patients had extended fasting periods, more swallowing assessments and interventions, longer hospitalization, and higher in-hospital mortality rate than non-AP patients. A total of 7% and 2% AP patients underwent video endoscopy and video fluorography respectively. In multivariate analysis, lower BMI, lower C-reactive protein, a lower ratio of homestay before hospitalization, a higher complication rate of cerebrovascular disease, dementia, and neuromuscular disease were noted as a characteristic of AP patients. Swallowing interventions were performed for 51% of the AP patients who had been hospitalized for more than two weeks. In univariate analysis, swallowing intervention improved in-hospital mortality. Lower AP recurrence before hospitalization and a lower ratio of homestay before hospitalization were indicated as characteristics of AP patients of the swallowing intervention group from multivariate analysis. Change in dietary pattern from normal to modified diet was observed more frequently in the swallowing intervention group. CONCLUSION: AP accounts for 38.4% of all pneumonia cases in acute care hospitals in Northern Japan. The use of swallowing evaluations and interventions, which may reduce the risk of dysphagia and may associate with lowering mortality in AP patients, is still not widespread.

Claudin expression during early postnatal development of the murine cochlea.

BACKGROUND: Claudins are major components of tight junctions, which form the paracellular barrier between the cochlear luminal and abluminal fluid compartments that supports the large transepithelial voltage difference and the large concentration differences of K+, Na+ and Ca2+ needed for normal cochlear function. Claudins are a family of more than 20 subtypes, but our knowledge about expression and localization of each subtype in the cochlea is limited. RESULTS: We examined by quantitative RT-PCR the expression of the mRNA of 24 claudin isoforms in mouse cochlea during postnatal development and localized the expression in separated fractions of the cochlea. Transcripts of 21 claudin isoforms were detected at all ages, while 3 isoforms (Cldn-16, - 17 and - 18) were not detected. Claudins that increased expression during development include Cldn-9, - 13, - 14, - 15, and -19v2, while Cldn-6 decreased. Those that do not change expression level during postnatal development include Cldn-1, - 2, - 3, - 4, - 5, - 7, - 8, -10v1, -10v2, - 11, - 12, -19v1, - 20, - 22, and - 23. Our investigation revealed unique localization of some claudins. In particular, Cldn-13 expression rapidly increases during early development and is mainly expressed in bone but only minimally in the lateral wall (including stria vascularis) and in the medial region (including the organ of Corti). No statistically significant changes in expression of Cldn-11, - 13, or - 14 were found in the cochlea of Slc26a4 -/- mice compared to Slc26a4 +/- mice. CONCLUSIONS: We demonstrated developmental patterns of claudin isoform transcript expression in the murine cochlea. Most of the claudins were associated with stria vascularis and organ of Corti, tissue fractions rich in tight junctions. However, this study suggests a novel function of Cldn-13 in the cochlea, which may be linked to cochlear bone marrow maturation.

[A Case Diagnosed as Iatrogenic Vesical Tuberculosis 4 Years after Intravesical Immunotherapy Using Bacillus Calmette-Guérin (BCG) for the Treatment of Bladder Carcinoma].

We report a case of iatrogenic vesical tuberculosis diagnosed 4 years after intravesical immunotherapy using Bacillus Calmette- Guérin (BCG) for the treatment of bladder carcinoma. A 72-year-old man underwent a transurethral resection (TUR) of multiple noninvasive urothelial carcinomas and intravesical BCG infusion (40 mg/week) for 7 weeks to prevent the recurrence of bladder carcinoma. BCG infusion therapy was terminated because of the appearance of Reiter's syndrome, including arthritis of the left toe joint, conjunctivitis and non-gonococcal urethritis as complications. The patient suffered from repeated cystitis, bladder atrophy and urethral stenosis. The cystitis improved with the administration of antibiotics (Levofloxacin) but persisted without a complete cure. Four years later, a cystoscopy revealed mucosal erosion and a white coating. An acid-fast bacteria examination of a urine sample using bacteria incubation and DNA PCR revealed the presence of Mycobacterium bovis. Finally, anti-tuberculosis therapy (INH＋REP＋EB) was initiated after the patient was diagnosed as having iatrogenic bladder tuberculosis resulting from BCG immunotherapy. The tuberculosis bacteria subsequently disappeared from the urine samples, and the gross appearance of the bladder mucosa improved. Bladder carcinoma has not recurred to date. Intravesical BCG infusion therapy has a good anti-tumor effect and can help prevent tumor recurrence after TUR therapy in case of noninvasive bladder carcinoma. However, there is a risk of severe complications arising from the BCG infusion. In the present case, an adequate bacteria examination was not performed, even though antibiotics were repeatedly administered for cystitis. In particular, the patient was not tested for the presence of acid-fast bacteria for 4 years after the intravesical BCG infusion therapy. Furthermore, among patients who received anti-bacteria therapy for repeated cystitis after BCG infusion, a bacteria examination including bacteria incubation, was not ordered in 19 out of 30 cases treated at our hospital over the past 5 years. In conclusion, bacteria examination, including tests for acid-fast bacteria, should be immediately performed when repeated and/or persistent cystitis occurs after BCG infusion therapy.

Outcomes of surgically treated rhinogenic optic neuropathy.

CONCLUSION: The visual outcomes of endoscopic treatment of optic neuropathy are favorable. However, no specific prognostic factor was associated with good outcome. OBJECTIVE: To investigate visual outcomes of patients with optic neuropathy caused by paranasal mucocele or sinusitis. METHODS: The clinical course in 38 patients with rhinogenic optic neuropathy was investigated retrospectively. All patients underwent endoscopic sinus surgery and ophthalmologists measured preoperative and postoperative visual acuities. RESULTS: Visual acuity completely recovered in 31 patients and partially in 2 patients, and did not recover in 5 patients. Age, sex, sinus involved (ethmoid or sphenoid), preoperative visual acuity, abnormal fundus findings, duration of symptoms, steroid use, and coexistence of diabetes mellitus showed no significant correlation with full recovery.

An analysis of clinical risk factors of deep neck infection.

OBJECTIVES: To clarify the clinical risk factors that aggravate deep neck infection. PATIENTS AND METHODS: Sixty-five patients with deep neck infection (abscess or cellulitis), 42 males and 23 females, who were treated at the ear, nose, and throat department in Iwaki Kyoritsu General Hospital in the past 10 years, were retrospectively reviewed. Cases of inflammation of the upper airway including the oral cavity, laryngopharynx, palate tonsil and salivary gland, and cases of lymphadenitis were investigated. These patients were divided into five localized types and one wide range type according to the abscess locations as follows: oral cavity floor type, upper deep cervical type, submandibular type, submental type, retropharyngeal type, and wide range type. RESULTS: Seventeen of the 65 patients had diabetes, and significantly more diabetics had the wide range type than the localized type (P<0.05, Fisher's test). Diabetes complication was more often seen in the upper deep cervical type among patients aged 61 years or older, and in the wide range type among males aged 41 years or older and elderly women aged 61 years or older. No patients with odontogenic infection or sialolithiasis had associated diabetes mellitus. Two cases developed mediastinitis, and one was caused by retrotonsillar abscess and needed thoracic drainage. More than half of the wide range type cases and more than a quarter of each of the localized type cases except the upper deep cervical type also had laryngeal edema, and eight of them needed emergency tracheotomy. Thirteen of the 40 cases had bacteria belonging to the Streptococcus milleri group (SMG), and all were detected in patients who underwent surgical drainage. Four of the 13 cases where SMG was detected showed drug resistance to some sorts of antibiotics. CONCLUSION: Oral disorders can develop deep neck infection independently of the presence of diabetes mellitus, compared with other causes. The presence of diabetes mellitus is associated with deep neck infection, aggravating parotitis and wide spread of inflammation. Retrotonsillar abscess often spreads to the retropharyngeal and parapharyngeal spaces, causing mediastinitis, so caution is necessary. Infection due to SMG tends to form abscess independently of diabetes mellitus. Since more than half of the wide range type and more than a quarter of each of the localized types except the upper deep cervical type were associated with laryngeal edema, airway management should be considered.

New treatment for invasive fungal sinusitis: three cases of chronic invasive fungal sinusitis treated with surgery and voriconazole.

Invasive fungal sinusitis is a relatively rare disease and can be divided into acute fulminant, chronic, and granulomatous invasive fungal sinusitis. The conventional treatment is radical surgery combined with systemic amphotericin B administration, but the poor prognosis and unestablished treatment options require a better therapeutic strategy. We report three cases of chronic invasive fungal sinusitis successfully treated with a combination of surgery and voriconazole, a new antifungal agent, with good responses in all patients. Voriconazole administration could form the basis for a new standard treatment for invasive fungal sinusitis.

Static and dynamic properties of phospholipid bilayer nanodiscs.

Nanodiscs are phospholipid-protein complexes which are relevant to nascent high-density lipoprotein and are applicable as a drug carrier and a tool to immobilize membrane proteins. We evaluated the structure and dynamics of the nanoparticles consisting of dimyristoylphosphatidylcholine (DMPC) and apolipoprotein A-I (apoA-I) with small-angle neutron scattering (SANS) and fluorescence methods and compared them with static/dynamic properties for large unilamellar vesicles. SANS revealed that the nanodisc includes a lipid bilayer with a thickness of 44 A and a radius of 37 A, in which each lipid occupies a smaller area than the reported molecular area of DMPC in vesicles. Fluorescence measurements suggested that DMPC possesses a lower entropy in nanodiscs than in vesicles, because apoA-I molecules, which surround the bilayer, force closer lipid packing, but allow water penetration to the acyl chain ends. Time-resolved SANS experiments revealed that nanodiscs represent a 20-fold higher lipid transfer via an entropically favorable process. The results put forward a conjunction of static/dynamic properties of nanodiscs, where the entropic constraints are responsible for the accelerated desorption of lipids.

Flip-flop of phospholipids in vesicles: kinetic analysis with time-resolved small-angle neutron scattering.

We applied a time-resolved small-angle neutron scattering technique to vesicle systems to determine interparticle transfer and flip-flop of phospholipids. Measurements were performed for large unilamellar vesicles, consisting of dimyristoylphosphatidylcholine (DMPC), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), or 1-palmitoyl-2-oleoylphosphatidic acid (POPA), which differ either in their acyl chains or headgroup. POPC, which is analogous to naturally occurring phosphatidylcholines, exhibited no transbilayer transfer and very slow interbilayer migration. POPC on the inner leaflet of vesicles did not flop even when phospholipase D converted all POPC molecules on the outer leaflet into POPA, which was shown to exhibit fast flip-flop. From these results, together with the observation that the flip-flop of DMPC was entirely inhibited in the presence of cholesterol, it is deduced that the flip-flop of phosphatidylcholines does not take place spontaneously in cellular plasma membranes rich in cholesterol and that it requires enzymatic activities of energy-dependent and/or -independent flippases/floppases.

Determination of interbilayer and transbilayer lipid transfers by time-resolved small-angle neutron scattering.

We applied a time-resolved small-angle neutron scattering technique to the vesicle system of dimyristoylphosphatidylcholine for the first time to determine lipid kinetics. The observed kinetics could be explicitly represented by a simple model that includes two independent kinetic parameters, i.e., the rates of transbilayer and interbilayer exchange. This technique is perfectly suited for the determination of lipid exchange kinetics in equilibrium and applicable to evaluation of the activity of the factors relevant to lipid migration, such as translocase and lipid transfer proteins.

[Management of cervical tuberculous lymphadenitis: evaluation of 6 cases].

Unlike other advanced nations, secondary spread of tuberculosis still occurs in Japan. Cervical tuberculous lymphadenitis is still an important disease of the neck, and between 2001 to 2005, we treated 6 patients with cervical tuberculous lymphadenitis. All 6 patients were females, and their ages ranged from 28 to 77 years old (average: 62 years). One patient had received antitubercular chemotherapy for pulmonary tuberculosis 40 years earlier. Two patients had a family history of pulmonary tuberculosis. One patient was an immigrant from Thailand. Three patients underwent open biopsy of the cervical lymph node, and were diagnosed with tuberculosis histologically. The remaining three patients had an abscess, and fine-needle aspiration (FNA) biopsy was performed. The diagnosis of tuberculosis was made by detection of acid-fast bacilli, MTD (Mycobacterium tuberculosis direct test), PCR (polymerase chain reaction), and culture. All six patients were treated with antitubercular chemotherapy for 6-9 months and recovered. MTD and PCR of the FNA sample seemed to enable early treatment. Attention needs to be paid to countries around Japan where tuberculosis is spreading. We suggest that treatment should be performed while at the same time making an effort to grasp the trend of spread in other countries as well as Japan.

A novel KCNQ4 one-base deletion in a large pedigree with hearing loss: implication for the genotype-phenotype correlation.

Autosomal-dominant, nonsyndromic hearing impairment is clinically and genetically heterogeneous. We encountered a large Japanese pedigree in which nonsyndromic hearing loss was inherited in an autosomal-dominant fashion. A genome-wide linkage study indicated linkage to the DFNA2 locus on chromosome 1p34. Mutational analysis of KCNQ4 encoding a potassium channel revealed a novel one-base deletion in exon 1, c.211delC, which generated a profoundly truncated protein without transmembrane domains (p.Q71fsX138). Previously, six missense mutations and one 13-base deletion, c.211_223del, had been reported in KCNQ4. Patients with the KCNQ4 missense mutations had younger-onset and more profound hearing loss than patients with the 211_223del mutation. In our current study, 12 individuals with the c.211delC mutation manifested late-onset and pure high-frequency hearing loss. Our results support the genotype-phenotype correlation that the KCNQ4 deletions are associated with later-onset and milder hearing impairment than the missense mutations. The phenotypic difference may be caused by the difference in pathogenic mechanisms: haploinsufficiency in deletions and dominant-negative effect in missense mutations.

The effects of hypoxia, premature birth, infection, ototoxic drugs, circulatory system and congenital disease on neonatal hearing loss.

OBJECTIVES: To investigate the incidence of neonatal hearing loss in well-baby populations and in a neonatal intensive care unit and to identify potential risk factors for hearing loss in a neonatal intensive care unit which the Joint Committee on Infant Hearing (JCIH) had not recommended. METHODS: Auditory screening was conducted in 226 infants (452 ears) born in Tohoku University from 2000 to 2001. The cases included 124 healthy newborn infants (248 ears), and 102 newborn infants (204 ears) treated in the neonatal intensive care unit (NICU). Hearing impairment was confirmed through a primary screening of the automated auditory brainstem response (AABR) and a secondary test of the auditory brainstem response (ABR) with otolaryngologic evaluation. Based on these examinations, we divided infants into two groups, 'Pass' and 'Refer'. RESULTS: Nine patients (15 ears) in Refer group were identified through our protocol. The incidence of the Refer group was 0.8% (1 out of 124) in the well-baby nursery, 7.8% (8 out of 102) in the NICU populations. The infants in Refer group were shown to have a higher incidence of congenital infection (P < 0.01), high C-reactive protein (CRP) (> or =10 mg/dl), chromosomal aberration, and central nervous system abnormality (P < 0.05). On the other hand, there were no statistical differences between the Pass and Refer groups in NICU, birth weight (<2200 g), gestational age, the values of total serum bilirubin, the values of arterial blood gases (pH, PaCO2 , PaO2 ), percutaneous oxygen saturation (SpO2), hemodynamics (blood pressure and heart rate) (P > 0.1). Respiratory status such as the Apgar score (the abbreviation for appearance, pulse, grimace, activity, respiration) (1 min; < or =4), (5 min; < or =6), Silverman retraction score, ototoxic drug use, respiratory distress syndrome (RDS), Meconium aspiration syndrome (MAS), and persistent pulmonary hypertension of newborn (PPHN) were also not statistically related to hearing loss (>0.999). CONCLUSION: Even in a small number of infants, there are positive relationships between hearing loss and congenital infection, high CRP (> or =10 mg/dl), chromosomal aberration and central nervous system abnormality. The CRP (> or =10 mg/dl) variable are not listed in the high-risk register published by the JCIH, but we can say that the variable may predict hearing impairment in our patient population. The possibility of autosomal recessive inheritance of genes for deafness is supposed when newborns have no other risk factors for hearing loss. This leads us to conclude that hearing screening is an effective way to find out hearing loss population.

Diastereoselective total synthesis of isocarbacyclin from L-ascorbic acid.

Diastereoselective total synthesis of isocarbacyclin, which features a fused bicyclic key intermediate available from l-ascorbic acid, is described. The key intermediate was prepared in multigram quantities by the Pauson-Khand reaction of l-ascorbic acid-based (R)-4,4-diallyl-2,2-dimethyl-5-(trimethylsilyl)ethynyl-1,3-dioxolane (3), discriminating diastereotopic groups and faces of the geminal allyl substituents.

Intramolecular catalytic Friedel-Crafts reactions with allenyl cations for the synthesis of quinolines and their analogues.

[reaction: see text] This paper describes a novel method to synthesize a quinoline backbone by incorporating allenyl cations into a catalytic intramolecular Friedel-Crafts reaction. The initial products were isomerized and aromatized upon treatment with acid and base, respectively, to give quinolines. The basic concept also proved to be promising for 1-benzazepine, 1-benzazocine, or isoquinoline synthesis.

Mutation detection of GJB2 using IsoCode and real-time quantitative polymerase chain reaction with SYBR green I dye for newborn hearing screening.

OBJECTIVES/HYPOTHESIS: Recent developments in molecular genetics have opened a new era in genetic analysis accompanied by new concepts concerning genetic disorders. Although 30 genes responsible for nonsyndromic deafness have been discovered as of March 27, 2003, the connexin 26 gene (GJB2) is commonly found in cases of deafness of unknown origins. The GJB2 contains a predicted open reading frame of 785 base pairs, which makes it relatively easy to detect mutations. Accordingly, mutation analysis of GJB2 should be suitable for the screening of congenital deafness. STUDY DESIGN: Prospective study. METHODS: IsoCode Stix is a useful device to isolate DNA from small samples of blood, which can be delivered from remote areas. To apply the detection of common mutations of GJB2 to hearing screening, DNA was extracted from several droplets of blood applied to the IsoCode device, and an allele-specific amplification method with real-time quantitative polymerase chain reaction was performed using GeneAmp 7700 with SYBR Green I dye. RESULTS: DNA extracted from IsoCode was purified within 45 minutes, which was sufficient to detect the full sequence of GJB2. Four types of common GJB2 mutations were reliably detected within 2.5 hours. CONCLUSION: IsoCode and real-time quantitative polymerase chain reaction will be promising tools for newborn screening of deafness genes in the future in DNA-based deafness screening, allowing early diagnosis of deafness and prompt training for language development.

Activity of an enzyme converting single-chain tissue-type plasminogen activator to the two-chain form in preovulatory human follicular fluid.

The biological role of the tissue-type plasminogen activator (tPA)/plasmin system has long been implicated in ovarian function. We have recently shown that the follicular fluid of human ovaries contains an alpha(2)-macroglobulin/protease complex capable of converting single-chain (sc) tPA to the two-chain (tc) enzyme tPA, suggesting the occurrence of its corresponding enzyme in a free form in the fluid. The aim of the current study is therefore to gain further information about the putative sctPA-converting enzyme present in follicular fluid. Incubation of human recombinant sctPA with the fluid brought about the production of tctPA. It was also demonstrated that tctPA production resulted in the activation of endogenous fluid plasminogen. Production of tctPA and plasmin both was strongly inhibited by aprotinin, suggesting that the enzyme is a serine protease. The sctPA-converting enzyme was partially purified from the fluid by column chromatographies. The enzyme preferably hydrolyzed synthetic peptide substrates containing arginine at the P(1) position. The enzyme preparation had a protease inhibitor profile similar to that observed with the crude fluid sample. These results clearly demonstrated that follicular fluid contains an enzyme capable of efficiently converting sctPA to tctPA. Discovery of this sctPA-converting enzyme strongly suggests that the tPA/plasmin system in the preovulatory follicle of human ovaries is operated through the proteolytic conversion of sctPA to tctPA rather than being regulated by a fibrin-dependent mechanism.

Catalytic alkynylation of ketones and aldehydes using quaternary ammonium hydroxide base.

Catalytic alkynylation of diverse ketones and aldehydes using nonmetallic benzyltrimethylammonium hydroxide or a basic resin of the hydroxide type in DMSO is described. Aliphatic or alicyclic carbonyl partners gave satisfactory results, whereas aromatic ones afforded products with low yields. When aromatic aldehydes were reacted with phenylacetylene, enones such as chalcone derivatives were obtained in place of ynols. These organobase-catalyzed systems provide a practical nonmetallic protocol for C[bond]C formation.

Transgenic expression of a dominant-negative connexin26 causes degeneration of the organ of Corti and non-syndromic deafness.

Hereditary deafness affects about 1 in 2000 children and mutations in the GJB2 gene are the major cause in various ethnic groups. GJB2 encodes connexin26, a putative channel component in cochlear gap junction. However, the pathogenesis of hearing loss caused by the GJB2 mutations remains obscure. The generation of a mouse model to study the function of connexin26 during hearing has been hampered by the fact that Gjb2 knockout mice are embryonic lethal. To establish viable model mice we generated transgenic mice expressing a mutant connexin26 with R75W mutation that was identified in a deaf family with autosomal-dominant inheritance. The previous expression analysis revealed that the R75W connexin26 inhibited the gap channel function of the co-expressed normal connexin26 in a dominant-negative fashion. We established two lines of transgenic mice that showed severe to profound hearing loss, deformity of supporting cells, failure in the formation of the tunnel of Corti and degeneration of sensory hair cells. Despite robust expression of the transgene, no obvious structural change was observed in the stria vascularis or spiral ligament that is rich in connexin26 and generates the endolymph. The high resting potential in cochlear endolymph essential for hair cell excitation was normally sustained. These results suggest that the GJB2 mutation disturbs homeostasis of cortilymph, an extracellular space surrounding the sensory hair cells, due to impaired K(+) transport by supporting cells, resulting in degradation of the organ of Corti, rather than affecting endolymph homeostasis in mice and probably in humans.