Fat Necrosis in Single Perforator Deep Inferior Epigastric Artery and Superficial Epigastric Artery Perforator Free Flaps: A Prospective Randomized Study.

Background: The deep inferior epigastric perforator (DIEP) flap is the standard of care in autologous breast reconstruction. The superficial inferior epigastric artery perforator flap (SIEA) is an alternative reconstructive option, with the compromise of less donor-site morbidity but variable perfusion to subscarpal fat zones. Fat necrosis is a known complication from marginal perfusion variability. Volumetric analysis of fat necrosis has not been performed between the two reconstructive options, nor has the amount of flap necrosis following radiation. Our objective was to compare rates and volume of fat necrosis between single-perforator DIEP and SIEA flap techniques. Methods: A single-center, blinded, prospective cohort study of patients randomized between SIEA and DIEP breast reconstruction was conducted over 2 years (June 2011-July 2013). Inclusion criteria were women undergoing immediate reconstruction following mastectomy. Randomization protocols were strictly followed. Fat necrosis volumetric analysis was determined by an ultrasound-trained attending surgeon at 12 months postoperatively. Patient demographics and adjuvant/neoadjuvant cancer treatment were analyzed. Statistical analyses included Mann-Whitney U tests, chi square, and/or Fisher exact tests. P values of 0.05 or less were considered significant. Results: Fat necrosis was detected in 11 of 46 flaps (23.9%), with a median area of 17.9 cm2. There was no significant difference in prevalence of fat necrosis between the two flap types (P = 0.19). Postoperative radiation did not increase the prevalence (P = 0.30) or extent (P = 0.92) of fat necrosis. Conclusion: Single-perforator DIEP and SIEA flaps have comparable rates of fat necrosis. Postoperative radiation did not result in increased prevalence or extent of fat necrosis.

Verrucous Carcinoma of the Foot: A Single Canadian Center Experience on Diagnosis, Management, and Outcomes.

INTRODUCTION: Verrucous carcinoma (VC) was first described in 1948 by Dr. Ackerman. It is a low-grade cutaneous squamous carcinoma that usually develops in the oral cavity, the anogenital region, and the plantar surface of the foot. Clinically, there is low suspicion for malignancy given the slow growth of VC lesions and their wart-like appearance. Diagnosis can be difficult because of the benign histological appearance with well-differentiated cells and absence of dysplasia. Surgical excision is the only satisfactory form of treatment for plantar VC; however, this becomes difficult given its benign clinical appearance and the pathologic misinterpretation of the lesion as a benign hyperplasia. While there are case reports and retrospective studies of patients with plantar VC in the literature, we present the largest case series of plantar VC within North America, with recurrence despite negative margins. METHODS: We report on all the plantar VC excised between 2014-2023. We report six cases of VC, their treatment, and their outcomes. RESULTS: Six patients obtained a diagnosis of plantar VC by incisional biopsy. All patients underwent excision of their lesions and had negative margins reported on the final pathology. All patients developed nonhealing wounds at the site of their lesion excision; therefore, biopsies were performed to confirm a recurrence. All patients had a recurrence of VC at the initial site. All patients underwent re-excision of the lesions. Despite negative margins again on final pathology, all patients had a subsequent second recurrence. Ultimately, all patients underwent an amputation as definitive management. Each patient had an average of 3 operations. There were 4 different surgeons and different pathologists reporting their findings. CONCLUSIONS: Our experience with plantar VC suggests that an aggressive approach to surgical management is needed. Furthermore, management is optimized with the combined expertise of an experienced dermatopathologist and surgeon. Despite negative margins and repeated excisions, VC lesions recur and invade local tissues to the extent that only amputation of the involved foot has resulted in cure.

Xylosyltransferase Bump-and-hole Engineering to Chemically Manipulate Proteoglycans in Mammalian Cells.

Mammalian cells orchestrate signalling through interaction events on their surfaces. Proteoglycans are an intricate part of these interactions, carrying large glycosaminoglycan polysaccharides that recruit signalling molecules. Despite their importance in development, cancer and neurobiology, a relatively small number of proteoglycans have been identified. In addition to the complexity of glycan extension, biosynthetic redundancy in the first protein glycosylation step by two xylosyltransferase isoenzymes XT1 and XT2 complicates annotation of proteoglycans. Here, we develop a chemical genetic strategy that manipulates the glycan attachment site of cellular proteoglycans. By employing a tactic termed bump- and-hole engineering, we engineer the two isoenzymes XT1 and XT2 to specifically transfer a chemically modified xylose analogue to target proteins. The chemical modification contains a bioorthogonal tag, allowing the ability to visualise and profile target proteins modified by both transferases in mammalian cells. The versatility of our approach allows pinpointing glycosylation sites by tandem mass spectrometry, and exploiting the chemical handle to manufacture proteoglycans with defined GAG chains for cellular applications. Engineered XT enzymes permit a view into proteoglycan biology that is orthogonal to conventional techniques in biochemistry.

Chemokine Oligomers and the Impact of Fondaparinux Binding.

Heparin, a widely used clinical anticoagulant, is generally well-tolerated; however, approximately 1% of patients develop heparin-induced thrombocytopenia (HIT), a serious side effect. While efforts to understand the role of chemokines in HIT development are ongoing, certain aspects remain less studied, such as the stabilization of chemokine oligomers by heparin. Here, we conducted a combined ion mobility-native mass spectrometry study to investigate the stability of chemokine oligomers and their complexes with fondaparinux, a synthetic heparin analog. Collision-induced dissociation and unfolding experiments provided clarity on the specificity and relevance of chemokine oligomers and their fondaparinux complexes with varying stoichiometries, as well as the stabilizing effects of fondaparinux binding.

Synovial Lipomatosis With Extra-articular Extension in the Arthritic Wrist: An Unexpected Diagnosis.

ABSTRACT: Synovial lipomatosis is a rare condition characterized by adipocyte proliferation within joint synovial tissue. It most commonly affects the knee and is typically intra-articular. Only 5 published case reports describe extra-articular synovial lipomatosis of the wrist. We present a case of a sexagenarian patient seen for his wrist arthropathy. His x-ray revealed pan-wrist arthritis and inflammatory soft tissue swelling. The patient was slated for a wrist fusion and Darrach procedure. Following the dorsal skin incision in the operating room, an unusual adipose mass was identified infiltrating all extensor compartments: midcarpal, radiocarpal, and distal radioulnar joints. The mass was excised and sent to pathology prior to proceeding with the slated surgery. Synovial lipomatosis was diagnosed postoperatively based on histopathology. Six weeks postoperatively, the wrist fusion had healed clinically and radiographically, and his pain had improved. There was no evidence of recurrence. Synovial lipomatosis is a rare entity that may imitate multiple other pathologies. It is possible that synovial lipomatosis may represent a secondary occurrence following degenerative articular disease or trauma in older patients. This is the first case report to date describing synovial lipomatosis of the wrist with extra-articular extension in the setting of pan-carpal wrist arthritis.

Structure and function of Semaphorin-5A glycosaminoglycan interactions.

Integration of extracellular signals by neurons is pivotal for brain development, plasticity, and repair. Axon guidance relies on receptor-ligand interactions crosstalking with extracellular matrix components. Semaphorin-5A (Sema5A) is a bifunctional guidance cue exerting attractive and inhibitory effects on neuronal growth through the interaction with heparan sulfate (HS) and chondroitin sulfate (CS) glycosaminoglycans (GAGs), respectively. Sema5A harbors seven thrombospondin type-1 repeats (TSR1-7) important for GAG binding, however the underlying molecular basis and functions in vivo remain enigmatic. Here we dissect the structural basis for Sema5A:GAG specificity and demonstrate the functional significance of this interaction in vivo. Using x-ray crystallography, we reveal a dimeric fold variation for TSR4 that accommodates GAG interactions. TSR4 co-crystal structures identify binding residues validated by site-directed mutagenesis. In vitro and cell-based assays uncover specific GAG epitopes necessary for TSR association. We demonstrate that HS-GAG binding is preferred over CS-GAG and mediates Sema5A oligomerization. In vivo, Sema5A:GAG interactions are necessary for Sema5A function and regulate Plexin-A2 dependent dentate progenitor cell migration. Our study rationalizes Sema5A associated developmental and neurological disorders and provides mechanistic insights into how multifaceted guidance functions of a single transmembrane cue are regulated by proteoglycans.

Glycoengineered keratinocyte library reveals essential functions of specific glycans for all stages of HSV-1 infection.

Viral and host glycans represent an understudied aspect of host-pathogen interactions, despite potential implications for treatment of viral infections. This is due to lack of easily accessible tools for analyzing glycan function in a meaningful context. Here we generate a glycoengineered keratinocyte library delineating human glycosylation pathways to uncover roles of specific glycans at different stages of herpes simplex virus type 1 (HSV-1) infectious cycle. We show the importance of cellular glycosaminoglycans and glycosphingolipids for HSV-1 attachment, N-glycans for entry and spread, and O-glycans for propagation. While altered virion surface structures have minimal effects on the early interactions with wild type cells, mutation of specific O-glycosylation sites affects glycoprotein surface expression and function. In conclusion, the data demonstrates the importance of specific glycans in a clinically relevant human model of HSV-1 infection and highlights the utility of genetic engineering to elucidate the roles of specific viral and cellular carbohydrate structures.

Chemokines form complex signals during inflammation and disease that can be decoded by extracellular matrix proteoglycans.

Chemokine-driven leukocyte recruitment is a key component of the immune response and of various diseases. Therapeutically targeting the chemokine system in inflammatory disease has been unsuccessful, which has been attributed to redundancy. We investigated why chemokines instead have specific, specialized functions, as demonstrated by multiple studies. We analyzed the expression of genes encoding chemokines and their receptors across species, tissues, and diseases. This analysis revealed complex expression patterns such that genes encoding multiple chemokines that mediated recruitment of the same leukocyte type were expressed in the same context, such as the genes encoding the CXCR3 ligands CXCL9, CXCL10, and CXCL11. Through biophysical approaches, we showed that these chemokines differentially interacted with extracellular matrix glycosaminoglycans (ECM GAGs), which was enhanced by sulfation of specific GAGs. Last, in vivo approaches demonstrated that GAG binding was critical for the CXCL9-dependent recruitment of specific T cell subsets but not of others, irrespective of CXCR3 expression. Our data demonstrate that interactions with ECM GAGs regulated whether chemokines were presented on cell surfaces or remained more soluble, thereby affecting chemokine availability and ensuring specificity of chemokine action. Our findings provide a mechanistic understanding of chemokine-mediated immune cell recruitment and identify strategies to target specific chemokines during inflammatory disease.

Direct observation of glycans bonded to proteins and lipids at the single-molecule level.

Proteins and lipids decorated with glycans are found throughout biological entities, playing roles in biological functions and dysfunctions. Current analytical strategies for these glycan-decorated biomolecules, termed glycoconjugates, rely on ensemble-averaged methods that do not provide a full view of positions and structures of glycans attached at individual sites in a given molecule, especially for glycoproteins. We show single-molecule analysis of glycoconjugates by direct imaging of individual glycoconjugate molecules using low-temperature scanning tunneling microscopy. Intact glycoconjugate ions from electrospray are soft-landed on a surface for their direct single-molecule imaging. The submolecular imaging resolution corroborated by quantum mechanical modeling unveils whole structures and attachment sites of glycans in glycopeptides, glycolipids, N-glycoproteins, and O-glycoproteins densely decorated with glycans.

Predicting Structural Motifs of Glycosaminoglycans using Cryogenic Infrared Spectroscopy and Random Forest.

In recent years, glycosaminoglycans (GAGs) have emerged into the focus of biochemical and biomedical research due to their importance in a variety of physiological processes. These molecules show great diversity, which makes their analysis highly challenging. A promising tool for identifying the structural motifs and conformation of shorter GAG chains is cryogenic gas-phase infrared (IR) spectroscopy. In this work, the cryogenic gas-phase IR spectra of mass-selected heparan sulfate (HS) di-, tetra-, and hexasaccharide ions were recorded to extract vibrational features that are characteristic to structural motifs. The data were augmented with chondroitin sulfate (CS) disaccharide spectra to assemble a training library for random forest (RF) classifiers. These were used to discriminate between GAG classes (CS or HS) and different sulfate positions (2-O-, 4-O-, 6-O-, and N-sulfation). With optimized data preprocessing and RF modeling, a prediction accuracy of >97% was achieved for HS tetra- and hexasaccharides based on a training set of only 21 spectra. These results exemplify the importance of combining gas-phase cryogenic IR ion spectroscopy with machine learning to improve the future analytical workflow for GAG sequencing and that of other biomolecules, such as metabolites.

Identification of global inhibitors of cellular glycosylation.

Small molecule inhibitors of glycosylation enzymes are valuable tools for dissecting glycan functions and potential drug candidates. Screening for inhibitors of glycosyltransferases are mainly performed by in vitro enzyme assays with difficulties moving candidates to cells and animals. Here, we circumvent this by employing a cell-based screening assay using glycoengineered cells expressing tailored reporter glycoproteins. We focused on GalNAc-type O-glycosylation and selected the GalNAc-T11 isoenzyme that selectively glycosylates endocytic low-density lipoprotein receptor (LDLR)-related proteins as targets. Our screen of a limited small molecule compound library did not identify selective inhibitors of GalNAc-T11, however, we identify two compounds that broadly inhibited Golgi-localized glycosylation processes. These compounds mediate the reversible fragmentation of the Golgi system without affecting secretion. We demonstrate how these inhibitors can be used to manipulate glycosylation in cells to induce expression of truncated O-glycans and augment binding of cancer-specific Tn-glycoprotein antibodies and to inhibit expression of heparan sulfate and binding and infection of SARS-CoV-2.

Chemokine CXCL4 interactions with extracellular matrix proteoglycans mediate widespread immune cell recruitment independent of chemokine receptors.

Leukocyte recruitment from the vasculature into tissues is a crucial component of the immune system but is also key to inflammatory disease. Chemokines are central to this process but have yet to be therapeutically targeted during inflammation due to a lack of mechanistic understanding. Specifically, CXCL4 (Platelet Factor 4, PF4) has no established receptor that explains its function. Here, we use biophysical, in vitro, and in vivo techniques to determine the mechanism underlying CXCL4-mediated leukocyte recruitment. We demonstrate that CXCL4 binds to glycosaminoglycan (GAG) sugars on proteoglycans within the endothelial extracellular matrix, resulting in increased adhesion of leukocytes to the vasculature, increased vascular permeability, and non-specific recruitment of a range of leukocytes. Furthermore, GAG sulfation confers selectivity onto chemokine localization. These findings present mechanistic insights into chemokine biology and provide future therapeutic targets.

Role of N-Glycosylation in FcγRIIIa interaction with IgG.

Immunoglobulins G (IgG) and their Fc gamma receptors (FcγRs) play important roles in our immune system. The conserved N-glycan in the Fc region of IgG1 impacts interaction of IgG with FcγRs and the resulting effector functions, which has led to the design of antibody therapeutics with greatly improved antibody-dependent cell cytotoxicity (ADCC) activities. Studies have suggested that also N-glycosylation of the FcγRIII affects receptor interactions with IgG, but detailed studies of the interaction of IgG1 and FcγRIIIa with distinct N-glycans have been hindered by the natural heterogeneity in N-glycosylation. In this study, we employed comprehensive genetic engineering of the N-glycosylation capacities in mammalian cell lines to express IgG1 and FcγRIIIa with different N-glycan structures to more generally explore the role of N-glycosylation in IgG1:FcγRIIIa binding interactions. We included FcγRIIIa variants of both the 158F and 158V allotypes and investigated the key N-glycan features that affected binding affinity. Our study confirms that afucosylated IgG1 has the highest binding affinity to oligomannose FcγRIIIa, a glycan structure commonly found on Asn162 on FcγRIIIa expressed by NK cells but not monocytes or recombinantly expressed FcγRIIIa.

Synthetic Heparan Sulfate Mimetic Pixatimod (PG545) Potently Inhibits SARS-CoV-2 by Disrupting the Spike-ACE2 Interaction.

Heparan sulfate (HS) is a cell surface polysaccharide recently identified as a coreceptor with the ACE2 protein for the S1 spike protein on SARS-CoV-2 virus, providing a tractable new therapeutic target. Clinically used heparins demonstrate an inhibitory activity but have an anticoagulant activity and are supply-limited, necessitating alternative solutions. Here, we show that synthetic HS mimetic pixatimod (PG545), a cancer drug candidate, binds and destabilizes the SARS-CoV-2 spike protein receptor binding domain and directly inhibits its binding to ACE2, consistent with molecular modeling identification of multiple molecular contacts and overlapping pixatimod and ACE2 binding sites. Assays with multiple clinical isolates of SARS-CoV-2 virus show that pixatimod potently inhibits the infection of monkey Vero E6 cells and physiologically relevant human bronchial epithelial cells at safe therapeutic concentrations. Pixatimod also retained broad potency against variants of concern (VOC) including B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617.2 (Delta), and B.1.1.529 (Omicron). Furthermore, in a K18-hACE2 mouse model, pixatimod significantly reduced SARS-CoV-2 viral titers in the upper respiratory tract and virus-induced weight loss. This demonstration of potent anti-SARS-CoV-2 activity tolerant to emerging mutations establishes proof-of-concept for targeting the HS-Spike protein-ACE2 axis with synthetic HS mimetics and provides a strong rationale for clinical investigation of pixatimod as a potential multimodal therapeutic for COVID-19.

Proteinopathy and Longitudinal Cognitive Decline in Parkinson Disease.

BACKGROUND AND OBJECTIVES: People with Parkinson disease (PD) commonly experience cognitive decline, which may relate to increased α-synuclein, tau, and ß-amyloid accumulation. This study examines whether the different proteins predict longitudinal cognitive decline in PD. METHODS: All participants (PD n = 152, controls n = 52) were part of a longitudinal study and completed a lumbar puncture for CSF protein analysis (α-synuclein, total tau [tau], and ß-amyloid42 [ß-amyloid]), a ß-amyloid PET scan, and/or provided a blood sample for APOE genotype (ε4+, ε4-), which is a risk factor for ß-amyloid accumulation. Participants also had comprehensive, longitudinal clinical assessments of overall cognitive function and dementia status, as well as cognitive testing of attention, language, memory, and visuospatial and executive function. We used hierarchical linear growth models to examine whether the different protein metrics predict cognitive change and multivariate Cox proportional hazard models to predict time to dementia conversion. Akaike information criterion was used to compare models for best fit. RESULTS: Baseline measures of CSF ß-amyloid predicted decline for memory (p = 0.04) and overall cognitive function (p = 0.01). APOE genotypes showed a significant group (ε4+, ε4-) effect such that ε4+ individuals declined faster than ε4- individuals in visuospatial function (p = 0.03). Baseline ß-amyloid PET significantly predicted decline in all cognitive measures (all p ≤ 0.004). Neither baseline CSF α-synuclein nor tau predicted cognitive decline. All 3 ß-amyloid--related metrics (CSF, PET, APOE) also predicted time to dementia. Models with ß-amyloid PET as a predictor fit the data the best. DISCUSSION: Presence or risk of ß-amyloid accumulation consistently predicted cognitive decline and time to dementia in PD. This suggests that ß-amyloid has high potential as a prognostic indicator and biomarker for cognitive changes in PD.

Molecular ontology of the parabrachial nucleus.

Diverse neurons in the parabrachial nucleus (PB) communicate with widespread brain regions. Despite evidence linking them to a variety of homeostatic functions, it remains difficult to determine which PB neurons influence which functions because their subpopulations intermingle extensively. An improved framework for identifying these intermingled subpopulations would help advance our understanding of neural circuit functions linked to this region. Here, we present the foundation of a developmental-genetic ontology that classifies PB neurons based on their intrinsic, molecular features. By combining transcription factor labeling with Cre fate-mapping, we find that the PB is a blend of two, developmentally distinct macropopulations of glutamatergic neurons. Neurons in the first macropopulation express Lmx1b (and, to a lesser extent, Lmx1a) and are mutually exclusive with those in a second macropopulation, which derive from precursors expressing Atoh1. This second, Atoh1-derived macropopulation includes many Foxp2-expressing neurons, but Foxp2 also identifies a subset of Lmx1b-expressing neurons in the Kölliker-Fuse nucleus (KF) and a population of GABAergic neurons ventrolateral to the PB ("caudal KF"). Immediately ventral to the PB, Phox2b-expressing glutamatergic neurons (some coexpressing Lmx1b) occupy the KF, supratrigeminal nucleus, and reticular formation. We show that this molecular framework organizes subsidiary patterns of adult gene expression (including Satb2, Calca, Grp, and Pdyn) and predicts output projections to the amygdala (Lmx1b), hypothalamus (Atoh1), and hindbrain (Phox2b/Lmx1b). Using this molecular ontology to organize, interpret, and communicate PB-related information could accelerate the translation of experimental findings from animal models to human patients.

Quantifying regional α -synuclein, amyloid ß, and tau accumulation in lewy body dementia.

OBJECTIVE: Parkinson disease (PD) is defined by the accumulation of misfolded α-synuclein (α-syn) in Lewy bodies and Lewy neurites. It affects multiple cortical and subcortical neuronal populations. The majority of people with PD develop dementia, which is associated with Lewy bodies in neocortex and referred to as Lewy body dementia (LBD). Other neuropathologic changes, including amyloid ß (Aß) and tau accumulation, occur in some LBD cases. We sought to quantify α-syn, Aß, and tau accumulation in neocortical, limbic, and basal ganglia regions. METHODS: We isolated insoluble protein from fresh frozen postmortem brain tissue samples for eight brains regions from 15 LBD, seven Alzheimer disease (AD), and six control cases. We measured insoluble α-syn, Aß, and tau with recently developed sandwich ELISAs. RESULTS: We detected a wide range of insoluble α-syn accumulation in LBD cases. The majority had substantial α-syn accumulation in most regions, and dementia severity correlated with neocortical α-syn. However, three cases had low neocortical levels that were indistinguishable from controls. Eight LBD cases had substantial Aß accumulation, although the mean Aß level in LBD was lower than in AD. The presence of Aß was associated with greater α-syn accumulation. Tau accumulation accompanied Aß in only one LBD case. INTERPRETATION: LBD is associated with insoluble α-syn accumulation in neocortical regions, but the relatively low neocortical levels in some cases suggest that other changes contribute to impaired function, such as loss of neocortical innervation from subcortical regions. The correlation between Aß and α-syn accumulation suggests a pathophysiologic relationship between these two processes.

State-of-the-art glycosaminoglycan characterization.

Glycosaminoglycans (GAGs) are heterogeneous acidic polysaccharides involved in a range of biological functions. They have a significant influence on the regulation of cellular processes and the development of various diseases and infections. To fully understand the functional roles that GAGs play in mammalian systems, including disease processes, it is essential to understand their structural features. Despite having a linear structure and a repetitive disaccharide backbone, their structural analysis is challenging and requires elaborate preparative and analytical techniques. In particular, the extent to which GAGs are sulfated, as well as variation in sulfate position across the entire oligosaccharide or on individual monosaccharides, represents a major obstacle. Here, we summarize the current state-of-the-art methodologies used for GAG sample preparation and analysis, discussing in detail liquid chromatograpy and mass spectrometry-based approaches, including advanced ion activation methods, ion mobility separations and infrared action spectroscopy of mass-selected species.

Dissecting structure-function of 3-O-sulfated heparin and engineered heparan sulfates.

Heparan sulfate (HS) polysaccharides are master regulators of diverse biological processes via sulfated motifs that can recruit specific proteins. 3-O-sulfation of HS/heparin is crucial for anticoagulant activity, but despite emerging evidence for roles in many other functions, a lack of tools for deciphering structure-function relationships has hampered advances. Here, we describe an approach integrating synthesis of 3-O-sulfated standards, comprehensive HS disaccharide profiling, and cell engineering to address this deficiency. Its application revealed previously unseen differences in 3-O-sulfated profiles of clinical heparins and 3-O-sulfotransferase (HS3ST)­specific variations in cell surface HS profiles. The latter correlated with functional differences in anticoagulant activity and binding to platelet factor 4 (PF4), which underlies heparin-induced thrombocytopenia, a known side effect of heparin. Unexpectedly, cells expressing the HS3ST4 isoenzyme generated HS with potent anticoagulant activity but weak PF4 binding. The data provide new insights into 3-O-sulfate structure-function and demonstrate proof of concept for tailored cell-based synthesis of next-generation heparins.

The interaction of chondroitin sulfate with a lipid monolayer observed by using nonlinear vibrational spectroscopy.

The first vibrational sum-frequency generation (VSFG) spectra of chondroitin sulfate (CS) interacting with dipalmitoyl phosphatidylcholine (DPPC) at air-liquid interface are reported here, collected at a laser repetition rate of 100 kHz. By studying the VSFG spectra in the regions of 1050-1450 cm-1, 2750-3180 cm-1, and 3200-3825 cm-1, it was concluded that in the presence of Ca2+ ions, the head groups together with the head-group-bound water molecules in the DPPC monolayer are strongly influenced by the interaction with CS, while the organization of the phospholipid tails remains mostly unchanged. The interactions were observed at a CS concentration below 200 nM, which exemplifies the potential of VSFG in studying biomolecular interactions at low physiological concentrations. The VSFG spectra recorded in the O-H stretching region at chiral polarization combination imply that CS molecules are organized into ordered macromolecular superstructures with a chiral secondary structure.