Biology subjects

Gargano, A.

Publications and source records attributed to Gargano, A..

5 recordsLinked to original sources

bioRxiv2026

Direct measurement and reconstruction of intact polyclonal IgG repertoires to preserve molecular and functional connectivity

Antibody function depends on the molecular pairing of antigen-binding Fab regions with Fc domains, linking antigen specificity to effector potential. However, antibody repertoire methods analyze these subunits separately, creating an inference problem in which Fc-Fab connectivity is lost. Here, we introduce a multi-level HPLC-MS workflow integrating native nanoflow cation-exchange chromatography-MS (nCEC-MS) of intact serum IgG with middle-up Fab and Fc/2 profiling. Native nCEC-MS reduces spectral congestion compared with denaturing reversed-phase HPLC-MS, enabling detection of 56-74 intact IgG mass features from 2 g of purified IgG per donor. Middle-up analysis provided complementary information on Fab diversity, subclass, and allotype, revealing donor-specific profiles. Reconstructing intact IgG1 masses from independently measured Fab and Fc/2 subunits partially recovers Fc-Fab molecular connectivity and supports interpretation of native intact mass features. Together, this framework provides an integrated view of the human IgG repertoire and establishes a foundation for studying its molecular topology in health and disease.

immunology↗

bioRxiv2026

Mapping the immune landscape in small cell lung cancer unveils a distinct tumor-reactive CD8+ T cell molecular signature

Small cell lung cancer (SCLC) is a highly aggressive malignancy with limited therapeutic advances. Unlike many other cancers, its immune landscape, particularly immune competence and T cell recognition, remains poorly characterized. Here, we generate a single-cell transcriptome atlas of the SCLC immune microenvironment with paired T cell receptor (TCR) sequencing. By linking T cell states with clonality and a multilayered functional screening, we identify 6 tumor-reactive TCRs that recognize and eradicate autologous SCLC cell lines. We delineate a novel SCLC-reactive CD8+ T cell signature (SCLC_TR), enabling the identification of 47 further SCLC-reactive TCRs. The SCLC_TR signature performs extremely well in pancreatic ductal adenocarcinoma (PDAC), another immune-cold tumor indication, and, most strikingly, patients with elevated SCLC_TR signature scores exhibited significantly improved survival, underlining its prognostic potential. Comparative cell-cell interaction analyses implicate several immunosuppressive mechanisms, with myeloid cells and CD4 regulatory T cells possibly acting as counterbalances to effector T cell activity in SCLC. In summary, our study challenges the prevailing notion of SCLC as an immune-cold tumor type by providing direct evidence of tumor-reactive T cell responses and introduces the SCLC_TR signature as a tool to identify tumor-specific T cells and their microenvironmental restraints and escape mechanisms, ultimately shaping next-generation immunotherapeutic strategies. O_FIG O_LINKSMALLFIG WIDTH=195 HEIGHT=200 SRC="FIGDIR/small/735200v1_ufig1.gif" ALT="Figure 1"> View larger version (69K): org.highwire.dtl.DTLVardef@1c25f2eorg.highwire.dtl.DTLVardef@1f6f519org.highwire.dtl.DTLVardef@553e57org.highwire.dtl.DTLVardef@6fe7e1_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2026

The volatile fatty-acid fingerprint of human fear

Human axillary odour can convey information about emotional state, but the molecular composition of fear related volatile emissions remains undefined. Here, we combine low background dual chamber axillary sampling, synchronous real time and offline mass spectrometry, chemical domain group independent component analysis and hierarchical Bayesian modelling to identify a molecular finger-print of acute fear during immersive virtual reality fear induction. In 37 healthy adults, this framework recovered five axillary volatile components associated with a continuous physiology derived fear index. The fingerprint comprised increased emissions of acetic, butyric, caproic and caprylic acids, octanal and acetone, together with decreased sulcatone, decanal, geranylacetone and citraconic anhydride. This coordinated pattern was reproducible across individuals and emerged from unsupervised chemical decomposition followed by regularised Bayesian selection, without prior biochemical constraints. Posterior predictive checks and leave one participant out refitting supported its robustness within the cohort. Its composition and directionality are consistent with sympathetic autonomic metabolic mobilisation, suggesting contributions from fatty acid handling, acetone related metabolism and axillary gland output. These findings define a candidate molecular cue of acute human fear and establish a replicable methodological template for decoding the chemical language of human emotion.

neuroscience↗

bioRxiv2021

Polyacrylamide co-N,N'-methylene bisacrylamide monoliths for high peak capacity HILIC-HRMS of intact proteins at low trifluoroacetic acid content

In this study, we optimized a polymerization mixture to synthesize polyacrylamide-co-N,N-methylene bisacrylamide monolithic stationary phases for hydrophilic-interaction chromatography (HILIC) of intact proteins. Thermal polymerization was performed, and the effects of varying the amount of crosslinker and the porogen composition on the separation performance of the resulting columns were studied. The homogeneity of the structure and the different porosities were examined through scanning electron microscopy. Further characterization of the monolithic structure revealed a permeable (Kf between 2.5 x 10-15 and 1.40 x 10-13 m2) and polar stationary phase suitable for HILIC. The HILIC separation performance of the different columns was assessed using gradient separation of a sample containing four intact proteins, with the best performing stationary phase exhibiting a peak capacity of 51 in a gradient of 25 min. Polyacrylamide-based materials were compared with a silica-based particulate amide phase (2.7 m core-shell particles). The monolith has no residual silanol sites and, therefore, fewer sites for ion-exchange interactions with proteins. Thus, it required lower concentrations of ion-pair reagent in HILIC of intact proteins. When using 0.1% of trifluoroacetic acid (TFA) the peak capacities of the two columns were similar (31 and 36 for the monolithic and packed column, respectively). However, when decreasing the concentration of TFA to 0.005%, the monolithic column maintained its separation performance and selectivity (peak capacity 26), whereas the packed column showed greatly reduced performance (peak capacity 7), lower selectivity, and inability to elute all four reference proteins. Finally, using a mobile phase containing 0.1% formic acid and 0.005% TFA the HILIC separation on the monolithic column was successfully hyphenated with high-resolution mass spectrometry. Detection sensitivity for protein and glycoproteins was increased and the amount of adducts formed was decreased in comparison with separations performed at 0.1% TFA.

biochemistry↗

bioRxiv2019

NGF-mediated photoablation of nociceptors reduces pain behavior in mice

Nerve growth factor (NGF) and its receptors TrkA and p75 play a key role in the development and function of peripheral nociceptive neurons. Here we describe novel technology to selectively photoablate TrkA positive nociceptors through delivery of a phototoxic agent coupled to an engineered NGF ligand and subsequent near infrared (NIR) illumination. We demonstrate that this approach allows for on demand and localized reversal of pain behaviors in mouse models of acute, inflammatory, neuropathic and joint pain. To target peripheral nociceptors we generated a SNAP-tagged NGF derivative, NGFR121W that binds to TrkA/p75 receptors but does not provoke signaling in TrkA positive cells or elicit pain behaviors in mice. NGFR121W-SNAP was coupled to the photosensitizer IRDye(R)700DX phthalocyanine (IR700) and injected subcutaneously. Following NIR illumination of the injected area, behavioral responses to nociceptive mechanical and sustained thermal stimuli, but not innocuous stimuli, were substantially reduced. Similarly, in models of inflammatory, osteoarthritic and neuropathic pain, mechanical hypersensitivity was abolished for three weeks following a single treatment regime. We demonstrate that this loss of pain behavior coincides with the retraction of neurons from the skin which then re-innervate the epidermis after 3 weeks corresponding with the return of mechanical hypersensitivity. Thus NGFR121W-SNAP-mediated photoablation is a minimally invasive approach to reversibly silence nociceptor input from the periphery, and control pain and hypersensitivity to mechanical stimuli.

neuroscience↗