Oral microbial colonization in laryngectomized patients as a possible cofactor of biofilm formation on their voice prostheses.

AIM: Biofilm formation on voice prostheses, which are used for voice rehabilitation in laryngectomized patients, is a main cause of device failure. The aim of this study was to assess whether the presence of periodontal pathogens in the biofilm on voice prostheses is related to that in the oral cavity and associated with the periodontal status of the patients. METHODS: Thirty-one laryngectomized patients were invited to participate, 13 of whom met exclusion criteria. The remaining 18 were classified according to the community periodontal index of treatment needs (community periodontal index of treatment needs (CPITN), grades 0-4). Biofilm samples from the oral cavity and voice prostheses were analysed by PCR-based hybridization for 11 pathogens. RESULTS: All dentate patients required periodontal treatment (CPITN-3: n = 4, CPITN-4: n = 8); the remaining six were edentulous. The diversity (i.e. number of bacterial species detected) of pathogens detected on the voice prostheses correlated significantly positively with the diversity of pathogens in the oral cavity and with clinical parameters. Furthermore, the diversity of pathogens differed significantly between dentate and edentulous patients. CONCLUSIONS: Results emphasize the oral cavity as an important source of bacteria for biofilm formation on voice prostheses. Whether these pathogens reduce the lifetime of the device by increased biofilm formation and/or increase the risk of silicone deterioration requires further study.

Advances in the Translation of Electrochemical Hydrogel-Based Sensors.

Novel biomaterials for bio- and chemical sensing applications have gained considerable traction in the diagnostic community with rising trends of using biocompatible and lowly cytotoxic material. Hydrogel-based electrochemical sensors have become a promising candidate for their swellable, nano-/microporous, and aqueous 3D structures capable of immobilizing catalytic enzymes, electroactive species, whole cells, and complex tissue models, while maintaining tunable mechanical properties in wearable and implantable applications. With advances in highly controllable fabrication and processability of these novel biomaterials, the possibility of bio-nanocomposite hydrogel-based electrochemical sensing presents a paradigm shift in the development of biocompatible, "smart," and sensitive health monitoring point-of-care devices. Here, recent advances in electrochemical hydrogels for the detection of biomarkers in vitro, in situ, and in vivo are briefly reviewed to demonstrate their applicability in ideal conditions, in complex cellular environments, and in live animal models, respectively, to provide a comprehensive assessment of whether these biomaterials are ready for point-of-care translation and biointegration. Sensors based on conductive and nonconductive polymers are presented, with highlights of nano-/microstructured electrodes that provide enhanced sensitivity and selectivity in biocompatible matrices. An outlook on current challenges that shall be addressed for the realization of truly continuous real-time sensing platforms is also presented.

Additively manufactured multiplexed electrochemical device (AMMED) for portable sample-to-answer detection.

Portable sample-to-answer devices with applications in point-of-care settings have emerged to obviate the necessity of centralized laboratories for biomarker analysis. In this work, a smartphone-operated and additively manufactured multiplexed electrochemical device (AMMED) is presented for the portable detection of biomarkers in blood and saliva. AMMED is comprised of a customized portable potentiostat with a multiplexing feature, a 3D-printed sample collection cartridge to handle three samples of saliva and blood at the same time, a smartphone application to remotely control the potentiostat, and a 3D-printed-based multiplexed microfluidic electrochemical biosensor (test chip). Here, by employing additive manufacturing techniques, a simple, cleanroom-free, and scalable approach was proposed for the fabrication of the test chip. Moreover, these techniques can bring about easy integration of AMMED components. Additionally, the test chip can be compatible with different affinity-based bioassays which can be implemented in a multiplexed manner for detection. The AMMED components were successfully characterized in terms of electrochemical and fluidic performance. Particularly, to demonstrate the biosensing capabilities of the device, the spike protein of the SARS-CoV-2 omicron variant and a well-established aptameric assay were selected as the representative biomarker and the bioassay, respectively. The proposed device accurately and selectively detected the target of interest in a rapid (5 min) and multiplex manner with a dynamic detection range of 1-10 000 pg ml-1 in different media, and the clinical feasibility was assessed by several saliva patient samples. AMMED offers a versatile sample-to-answer platform that can be used for the detection of various biomarkers present in biofluids.

Familial hemophagocytic lymphohistiocytosis type 5 (FHL-5) is caused by mutations in Munc18-2 and impaired binding to syntaxin 11.

Rapid intracellular transport and secretion of cytotoxic granules through the immunological synapse requires a balanced interaction of several proteins. Disturbance of this highly regulated process underlies familial hemophagocytic lymphohistiocytosis (FHL), a genetically heterogeneous autosomal-recessive disorder characterized by a severe hyperinflammatory phenotype. Here, we have assigned FHL-5 to a 1 Mb region on chromosome 19p by using high-resolution SNP genotyping in eight unrelated FHL patients from consanguineous families. Subsequently, we found nine different mutations, either truncating or missense, in STXBP2 in twelve patients from Turkey, Saudi Arabia, and Central Europe. STXBP2 encodes syntaxin binding protein 2 (Munc18-2), involved in the regulation of vesicle transport to the plasma membrane. We have identified syntaxin 11, a SNARE protein mutated in FHL-4, as an interaction partner of STXBP2. This interaction is eliminated by the missense mutations found in our FHL-5 patients, which leads to a decreased stability of both proteins, as shown in patient lymphocytes. Activity of natural killer and cytotoxic T cells was markedly reduced or absent, as determined by CD107 degranulation. Our findings thus identify a key role for STXBP2 in lytic granule exocytosis.

A Versatile Biomimic Nanotemplating Fluidic Assay for Multiplex Quantitative Monitoring of Viral Respiratory Infections and Immune Responses in Saliva and Blood.

The last pandemic exposed critical gaps in monitoring and mitigating the spread of viral respiratory infections at the point-of-need. A cost-effective multiplexed fluidic device (NFluidEX), as a home-test kit analogous to a glucometer, that uses saliva and blood for parallel quantitative detection of viral infection and body's immune response in an automated manner within 11 min is proposed. The technology integrates a versatile biomimetic receptor based on molecularly imprinted polymers in a core-shell structure with nano gold electrodes, a multiplexed fluidic-impedimetric readout, built-in saliva collection/preparation, and smartphone-enabled data acquisition and interpretation. NFluidEX is validated with Influenza A H1N1 and SARS-CoV-2 (original strain and variants of concern), and achieves low detection limit in saliva and blood for the viral proteins and the anti-receptor binding domain (RBD) Immunoglobulin G (IgG) and Immunoglobulin M (IgM), respectively. It is demonstrated that nanoprotrusions of gold electrodes are essential for the fine templating of antibodies and spike proteins during molecular imprinting, and differentiation of IgG and IgM in whole blood. In the clinical setting, NFluidEX achieves 100% sensitivity and 100% specificity by testing 44 COVID-positive and 25 COVID-negative saliva and blood samples on par with the real-time quantitative polymerase chain reaction (p < 0.001, 95% confidence) and the enzyme-linked immunosorbent assay.

Improving Access to Lactation Consultation and Early Breast Milk Use in an Outborn NICU.

INTRODUCTION: Increasing the use of breast milk in critically ill neonates is an important priority to improve neonatal care. Lactation consultants (LCs) educate mothers about evidence-based benefits of breast milk and provide technical support. LC support can lead to increased breastfeeding initiation. The project aim was to improve access to lactation services for mothers of patients admitted at <48 hours after birth to an exclusively outborn level III/IV neonatal intensive care unit (NICU). METHODS: The interventions included (1) implementation of an automatic electronic admission order for a lactation consult, (2) initiation of a daily lactation team notification, (3) assignment of a consistent NICU LC, and (4) targeted education. The percent of mothers who received lactation consults, the time to the first consultation, and the percent of patients receiving breast milk at 7 days of age were measured over 32 months and analyzed using statistical process control charts. RESULTS: The lactation consultation rate increased significantly from 74% to 88% with a shift in the mean by statistical process control chart analysis that was sustained over time. Concurrently, the time to first lactation consultation significantly decreased from hospital days 5 to 3.3, and variation decreased. Rates of breast milk use at 7 days of age also significantly increased from 75.6% to 89.6%. CONCLUSIONS: Targeted quality improvement interventions led to an increased rate of lactation consultations, decreased time to first lactation consult, and increased rate of breast milk use at 7 days of age. These interventions could feasibly be implemented in similar referral NICU settings.

Lineage-specific control of TFIIH by MITF determines transcriptional homeostasis and DNA repair.

The melanocytic lineage, which is prominently exposed to ultraviolet radiation (UVR) and radiation-independent oxidative damage, requires specific DNA-damage response mechanisms to maintain genomic and transcriptional homeostasis. The coordinate lineage-specific regulation of intricately intertwined DNA repair and transcription is incompletely understood. Here we demonstrate that the Microphthalmia-associated transcription factor (MITF) directly controls general transcription and UVR-induced nucleotide excision repair by transactivation of GTF2H1 as a core element of TFIIH. Thus, MITF ensures the rapid resumption of transcription after completion of strand repair and maintains transcriptional output, which is indispensable for survival of the melanocytic lineage including melanoma in vitro and in vivo. Moreover, MITF controls c-MYC implicated in general transcription by transactivation of far upstream binding protein 2 (FUBP2/KSHRP), which induces c-MYC pulse regulation through TFIIH, and experimental depletion of MITF results in consecutive loss of CDK7 in the TFIIH-CAK subcomplex. Targeted for proteasomal degradation, CDK7 is dependent on transactivation by MITF or c-MYC to maintain a steady state. The dependence of TFIIH-CAK on sequence-specific MITF and c-MYC constitutes a previously unrecognized mechanism feeding into super-enhancer-driven or other oncogenic transcriptional circuitries, which supports the concept of a transcription-directed therapeutic intervention in melanoma.

Combined inhibition of receptor tyrosine and p21-activated kinases as a therapeutic strategy in childhood ALL.

Receptor tyrosine kinase (RTK)-dependent signaling has been implicated in the pathogenesis of acute lymphoblastic leukemia (ALL) of childhood. However, the RTK-dependent signaling state and its interpretation with regard to biological behavior are often elusive. To decipher signaling circuits that link RTK activity with biological output in vivo, we established patient-derived xenograft ALL (PDX-ALL) models with dependencies on fms-like tyrosine kinase 3 (FLT3) and platelet-derived growth factor receptor ß (PDGFRB), which were interrogated by phosphoproteomics using iTRAQ mass spectrometry. Signaling circuits were determined by receptor type and cellular context with few generic features, among which we identified group I p21-activated kinases (PAKs) as potential therapeutic targets. Growth factor stimulation markedly increased catalytic activities of PAK1 and PAK2. RNA interference (RNAi)-mediated or pharmacological inhibition of PAKs using allosteric or adenosine triphosphate (ATP)-competitive compounds attenuated cell growth and increased apoptosis in vitro. Notably, PAK1- or PAK2-directed RNAi enhanced the antiproliferative effects of the type III RTK and protein kinase C inhibitor midostaurin. Treatment of FLT3- or PDGFRB-dependent ALLs with ATP-competitive PAK inhibitors markedly decreased catalytic activities of both PAK isoforms. In FLT3-driven ALL, this effect was augmented by coadministration of midostaurin resulting in synergistic effects on growth inhibition and apoptosis. Finally, combined treatment of FLT3 D835H PDX-ALL with the ATP-competitive group I PAK inhibitor FRAX486 and midostaurin in vivo significantly prolonged leukemia progression-free survival compared with midostaurin monotherapy or control. Our study establishes PAKs as potential downstream targets in RTK-dependent ALL of childhood, the inhibition of which might help prevent the selection or acquisition of resistance mutations toward tyrosine kinase inhibitors.

Leukemia-associated mutations in SHIP1 inhibit its enzymatic activity, interaction with the GM-CSF receptor and Grb2, and its ability to inactivate PI3K/AKT signaling.

The inositol 5-phosphatase SHIP1 is a negative regulator of the PI3K/AKT pathway, which is constitutively activated in 50-70% of acute myeloid leukemias (AML). Ten different missense mutations in SHIP1 have been described in 3% of AML patients suggesting a functional role of SHIP1 in AML. Here, we report the identification of two new SHIP1 mutations T162P and R225W that were detected in 2 and 1 out of 96 AML patients, respectively. The functional analysis of all 12 AML-associated SHIP1 mutations, one ALL-associated SHIP1 mutation (Q1076X) and a missense SNP (H1168Y) revealed that two mutations i.e. Y643H and P1039S abrogated the ability of SHIP1 to reduce constitutive PI3K/AKT signaling in Jurkat cells. The loss of function of SHIP1 mutant Y643H which is localized in the inositol phosphatase domain was due to a reduction of the specific activity by 84%. Because all other SHIP1 mutants had a normal enzymatic activity, we assumed that these SHIP1 mutants may be functionally impaired due to a loss of interaction with plasma membrane receptors or adapter proteins. In agreement with this model, we found that the SHIP1 mutant F28L located in the FLVR motif of the SH2 domain was incapable of binding tyrosine-phosphorylated proteins including the GM-CSF receptor and that the SHIP1 mutant Q1076X lost its ability to bind to the C-terminal SH3 domain of the adapter protein Grb2. In addition, SHIP1 mutant P1039S which does not reduce PI3K/AKT signaling anymore is located in a PXXP SH3 domain consensus binding motif suggesting that mutation of the conserved proline residue interferes with binding of SHIP1 to a so far unidentified SH3 domain containing protein. In summary, our data indicate that SHIP1 mutations detected in human leukemia patients impair the negative regulatory function of SHIP1 on PI3K/AKT signaling in leukemia cells either directly by reduced enzymatic activity or indirectly by disturbed protein interaction with tyrosine-phosphorylated membrane receptors or adapter proteins. These results further support a functional role of SHIP1 as tumor suppressor protein in the pathogenesis of AML.