Klebsiella pneumoniae O-polysaccharide biosynthesis highlights the diverse organization of catalytic modules in ABC transporter-dependent glycan assembly.

Klebsiella pneumoniae provides influential prototypes for lipopolysaccharide O antigen (OPS) biosynthesis in Gram-negative bacteria. Sequences of OPS-biosynthesis gene clusters in serotypes O4 and O7 suggest fundamental differences in the organization of required enzyme modules compared to other serotypes. Furthermore, some required activities were not assigned by homology shared with characterized enzymes. The goal of this study was therefore to resolve the serotype O4 and O7 pathways to expand our broader understanding of glycan polymerization and chain termination processes. The O4 and O7 antigens were produced from cloned genetic loci in recombinant Escherichia coli. Systematic in vivo and in vitro approaches were then applied to assign each enzyme in each of the pathways, defining the necessary components for polymerization and chain termination. OPS assembly is accomplished by multiprotein complexes formed by interactions between polymerase components variably distributed in single and multimodule proteins. In each complex, a terminator function is present in a protein containing a characteristic coiled-coil molecular ruler, which determines glycan chain length. In serotype O4, we discovered a CMP-α-3-deoxy-ᴅ-manno-octulosonic acid-dependent chain-terminating glycosyltransferase that is the founding member of a new glycosyltransferase family (GT137) and potentially identifies a new glycosyltransferase fold. The O7 OPS is terminated by a methylphosphate moiety, like the K. pneumoniae O3 antigen, but the methyltransferase-kinase enzyme pairs responsible for termination in these serotypes differ in sequence and predicted structures. Together, the characterization of O4 and O7 has established unique enzyme activities and provided new insight into glycan-assembly strategies that are widely distributed in bacteria.

SARS-CoV-2 infection of airway cells causes intense viral and cell shedding, two spreading mechanisms affected by IL-13.

Muco-obstructive lung diseases are typically associated with high risks of COVID-19 severity; however, allergic asthma showed reduced susceptibility. To investigate viral spread, primary human airway epithelial (HAE) cell cultures were infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and host­virus interactions were examined via electron microscopy, immunohistochemistry, RNA in situ hybridization, and gene expression analyses. In HAE cell cultures, angiotensin-converting enzyme 2 (ACE2) expression governed cell tropism and viral load and was up-regulated by infection. Electron microscopy identified intense viral egress from infected ciliated cells and severe cytopathogenesis, culminating in the shedding of ciliated cells packed with virions, providing a large viral reservoir for spread and transmission. Intracellular stores of MUC5AC, a major airway mucin involved in asthma, were rapidly depleted, likely to trap viruses. To mimic asthmatic airways, HAE cells were treated with interleukin-13 (IL-13), which reduced viral titers, viral messenger RNA, and cell shedding, and significantly diminished the number of infected cells. Although mucus hyperproduction played a shielding role, IL-13­treated cells maintained a degree of protection despite the removal of mucus. Using Gene Expression Omnibus databases, bulk RNA-sequencing analyses revealed that IL-13 up-regulated genes controlling glycoprotein synthesis, ion transport, and antiviral processes (albeit not the typical interferon-induced genes) and down-regulated genes involved in cilial function and ribosomal processing. More precisely, we showed that IL-13 reduced ACE2 expression, intracellular viral load, and cell-to-cell transmission while increasing the cilial keratan sulfate coating. In conclusion, intense viral and cell shedding caused by SARS-CoV-2 infection was attenuated by IL-13, which affected viral entry, replication, and spread.

A neutralizing antibody target in early HIV-1 infection was recapitulated in rhesus macaques immunized with the transmitted/founder envelope sequence.

Transmitted/founder (T/F) HIV-1 envelope proteins (Envs) from infected individuals that developed neutralization breadth are likely to possess inherent features desirable for vaccine immunogen design. To explore this premise, we conducted an immunization study in rhesus macaques (RM) using T/F Env sequences from two human subjects, one of whom developed potent and broad neutralizing antibodies (Z1800M) while the other developed little to no neutralizing antibody responses (R66M) during HIV-1 infection. Using a DNA/MVA/protein immunization protocol, 10 RM were immunized with each T/F Env. Within each T/F Env group, the protein boosts were administered as either monomeric gp120 or stabilized trimeric gp140 protein. All vaccination regimens elicited high titers of antigen-specific IgG, and two animals that received monomeric Z1800M Env gp120 developed autologous neutralizing activity. Using early Env escape variants isolated from subject Z1800M as guides, the serum neutralizing activity of the two immunized RM was found to be dependent on the gp120 V5 region. Interestingly, the exact same residues of V5 were also targeted by a neutralizing monoclonal antibody (nmAb) isolated from the subject Z1800M early in infection. Glycan profiling and computational modeling of the Z1800M Env gp120 immunogen provided further evidence that the V5 loop is exposed in this T/F Env and was a dominant feature that drove neutralizing antibody targeting during infection and immunization. An expanded B cell clonotype was isolated from one of the neutralization-positive RM and nmAbs corresponding to this group demonstrated V5-dependent neutralization similar to both the RM serum and the human Z1800M nmAb. The results demonstrate that neutralizing antibody responses elicited by the Z1800M T/F Env in RM converged with those in the HIV-1 infected human subject, illustrating the potential of using immunogens based on this or other T/F Envs with well-defined immunogenicity as a starting point to drive breadth.

The biosynthetic origin of ribofuranose in bacterial polysaccharides.

Bacterial surface polysaccharides are assembled by glycosyltransferase enzymes that typically use sugar nucleotide or polyprenyl-monophosphosugar activated donors. Characterized representatives exist for many monosaccharides but neither the donor nor the corresponding glycosyltransferases have been definitively identified for ribofuranose residues found in some polysaccharides. Klebsiella pneumoniae O-antigen polysaccharides provided prototypes to identify dual-domain ribofuranosyltransferase proteins catalyzing a two-step reaction sequence. Phosphoribosyl-5-phospho-D-ribosyl-α-1-diphosphate serves as the donor for a glycan acceptor-specific phosphoribosyl transferase (gPRT), and a more promiscuous phosphoribosyl-phosphatase (PRP) then removes the residual 5'-phosphate. The 2.5-Å resolution crystal structure of a dual-domain ribofuranosyltransferase ortholog from Thermobacillus composti revealed a PRP domain that conserves many features of the phosphatase members of the haloacid dehalogenase family, and a gPRT domain that diverges substantially from all previously characterized phosphoribosyl transferases. The gPRT represents a new glycosyltransferase fold conserved in the most abundant ribofuranosyltransferase family.

Markov blankets: Realism and our ontological commitments.

The authors argue that their target is orthogonal to the realism and instrumentalist debate. I argue that it is born directly from it. While the distinction is helpful in illuminating how some ontological commitments demand a theory of implementation, it's less clear whether different views cleanly map onto the epistemic and metaphysical uses defined in the paper.

Lipopolysaccharide O-antigens-bacterial glycans made to measure.

Lipopolysaccharides are critical components of bacterial outer membranes. The more conserved lipid A part of the lipopolysaccharide molecule is a major element in the permeability barrier imposed by the outer membrane and offers a pathogen-associated molecular pattern recognized by innate immune systems. In contrast, the long-chain O-antigen polysaccharide (O-PS) shows remarkable structural diversity and fulfills a range of functions, depending on bacterial lifestyles. O-PS production is vital for the success of clinically important Gram-negative pathogens. The biological properties and functions of O-PSs are mostly independent of specific structures, but the size distribution of O-PS chains is particularly important in many contexts. Despite the vast O-PS chemical diversity, most are produced in bacterial cells by two assembly strategies, and the different mechanisms employed in these pathways to regulate chain-length distribution are emerging. Here, we review our current understanding of the mechanisms involved in regulating O-PS chain-length distribution and discuss their impact on microbial cell biology.

The Amyloid Fibril-Forming ß-Sheet Regions of Amyloid ß and α-Synuclein Preferentially Interact with the Molecular Chaperone 14-3-3ζ.

14-3-3 proteins are abundant, intramolecular proteins that play a pivotal role in cellular signal transduction by interacting with phosphorylated ligands. In addition, they are molecular chaperones that prevent protein unfolding and aggregation under cellular stress conditions in a similar manner to the unrelated small heat-shock proteins. In vivo, amyloid ß (Aß) and α-synuclein (α-syn) form amyloid fibrils in Alzheimer's and Parkinson's diseases, respectively, a process that is intimately linked to the diseases' progression. The 14-3-3ζ isoform potently inhibited in vitro fibril formation of the 40-amino acid form of Aß (Aß40) but had little effect on α-syn aggregation. Solution-phase NMR spectroscopy of 15N-labeled Aß40 and A53T α-syn determined that unlabeled 14-3-3ζ interacted preferentially with hydrophobic regions of Aß40 (L11-H21 and G29-V40) and α-syn (V3-K10 and V40-K60). In both proteins, these regions adopt ß-strands within the core of the amyloid fibrils prepared in vitro as well as those isolated from the inclusions of diseased individuals. The interaction with 14-3-3ζ is transient and occurs at the early stages of the fibrillar aggregation pathway to maintain the native, monomeric, and unfolded structure of Aß40 and α-syn. The N-terminal regions of α-syn interacting with 14-3-3ζ correspond with those that interact with other molecular chaperones as monitored by in-cell NMR spectroscopy.

The assembly of (+)-vincadifformine- and (-)-tabersonine-derived monoterpenoid indole alkaloids in Catharanthus roseus involves separate branch pathways.

The biological activity of monoterpenoid indole alkaloids (MIAs) has led to their use in cancer treatment and other medical applications. Their biosynthesis has involved the formation of reactive intermediates by responsible enzymes to elaborate several different chemical scaffolds. Modification of scaffolds through different substitution reactions has produced chemically diverse MIAs and related biological activities. The present study characterizes the three-step pathway involved in the formation of (+)-echitovenine, the major O-acetylated MIA of Catharanthus roseus roots, and differentiates it from a parallel pathway involved in the formation of hörhammericine. Separate hydrolases convert a common reactive MIA intermediate to aspidosperma skeletons of opposite specific rotations, that is (+)-vincadifformine and (-)-tabersonine, respectively. The formation of (+) minovincinine from (+) vincadifformine 19-hydroxylase (V19H) is catalyzed by a root-specific cytochrome P450 with high amino acid sequence similarity to the leaf-specific tabersonine-3-hydroxylase involved in vindoline biosynthesis. Similarly, O-acetylation of (+)-minovincinine to form (+) echitovenine involves minovincinine-O-acetytransferase. The substrate specificity of V19H and MAT for their respective (+)-enantiomers defines the separate enantiomer-specific pathway involved in (+)-echitovenine biosynthesis and differentiates it from a parallel (-)-enantiomer-specific pathway involved in the formation of hörhammericine from (-)-tabersonine.

Somatic mismatch repair testing in evaluation of Lynch syndrome: The gap between preferred and current practices.

Lynch syndrome (LS) is a hereditary cancer predisposition syndrome primarily defined by increased risk for colorectal and uterine cancers. Individuals with germline pathogenic variants in the mismatch repair (MMR) genes (MLH1, MSH2/EPCAM, MSH6, and PMS2) are diagnosed with LS and recommended high-risk screening protocols to increase prevention and early detection of LS-related cancers. Tumor testing can help identify those at high risk for LS, but sometimes creates uncertainty with discordant screening and germline results, or unexplained mismatch repair deficiency (UMMRD). Somatic testing for MMR genes may help resolve UMMRD, potentially clarifying LS status and modifying cancer surveillance. However, guidelines for such testing are currently limited. This survey of cancer genetic counselors (GCs) aimed to examine current versus preferred ordering practices and interpretation of somatic MMR testing results in LS evaluation. Two hundred eligible GCs practicing in the United States and Canada were recruited from the National Society of Genetic Counselors. Participants answered questions regarding ordering practices, barriers to somatic MMR testing, theoretical scenarios, and desire for further guidelines. Statistical analysis was performed using chi-square, Fisher's exact, and Wilcoxon rank-sum tests, while themes were identified from free-text responses. Most respondents did not include somatic MMR testing in the LS work-up, despite three-quarters reporting they were 'somewhat comfortable' or 'extremely comfortable' with interpreting these results. Approximately half of participants indicated interest in ordering concurrent somatic MMR and germline testing for each of the four theoretical scenarios. Over three-quarters of individuals reported barriers to ordering somatic MMR testing, with cost and coordinating tissue samples most commonly cited. The frequently reported laboratory- and insurance-related barriers may contribute to the gap between preferred and current ordering practices for somatic MMR testing. Nearly all respondents endorsed additional guidelines for this testing, which could reduce barriers and inform screening recommendations for patients with UMMRD and their family members.

Single polysaccharide assembly protein that integrates polymerization, termination, and chain-length quality control.

Lipopolysaccharides (LPS) are essential outer membrane glycolipids in most gram-negative bacteria. Biosynthesis of the O-antigenic polysaccharide (OPS) component of LPS follows one of three widely distributed strategies, and similar processes are used to assemble other bacterial surface glycoconjugates. This study focuses on the ATP-binding cassette (ABC) transporter-dependent pathway, where glycans are completed on undecaprenyl diphosphate carriers at the cytosol:membrane interface, before export by the ABC transporter. We describe Raoultella terrigena WbbB, a prototype for a family of proteins that, remarkably, integrates several key activities in polysaccharide biosynthesis into a single polypeptide. WbbB contains three glycosyltransferase (GT) modules. Each of the GT102 and GT103 modules characterized here represents a previously unrecognized GT family. They form a polymerase, generating a polysaccharide of [4)-α-Rhap-(1→3)-ß-GlcpNAc-(1→] repeat units. The polymer chain is terminated by a ß-linked Kdo (3-deoxy-d-manno-oct-2-ulosonic acid) residue added by a third GT module belonging to the recently discovered GT99 family. The polymerase GT modules are separated from the GT99 chain terminator by a coiled-coil structure that forms a molecular ruler to determine product length. Different GT modules in the polymerase domains of other family members produce diversified OPS structures. These findings offer insight into glycan assembly mechanisms and the generation of antigenic diversity as well as potential tools for glycoengineering.

Ultrasound-Guided Biceps Tendon Sheath Injection: Spectrum of Preprocedure Appearances.

OBJECTIVES: To determine the frequency of ultrasound (US) appearances of the extra-articular long head of the proximal biceps tendon in patients referred for US-guided biceps tendon sheath injections. METHODS: We reviewed our US-guided biceps tendon sheath injections between January 2015 and December 2017, noting the appearance of the biceps tendon and sheath, as well as the needle size used, and what was injected. Clinical electronic medical records were also reviewed to determine safety and patients' responses. RESULTS: A total of 300 US-guided biceps tendon sheath injections were performed for anterior shoulder pain. Preliminary US evaluations revealed that 129 of 300 (43%) patients had a normal US appearance of the biceps tendon; 110 (36.6%) had tendinosis; 13 (4.3%) had tenosynovitis; 31 (10.3%) had both tendinosis and tenosynovitis; 8 (2.7%) had a biceps tendon tear; and 9 (3%) had a history of a tenodesis. Of 81 patients who had pain relief after the injection, 41 had a normal tendon appearance on US, and 40 had an abnormal US appearance. CONCLUSIONS: A large minority of patients with anterior shoulder pain clinically suspected to be due to the biceps tendon have a normal-appearing tendon and sheath. This should not dissuade the operator from performing the procedure.

Short Total Synthesis of Ajoene.

We describe a short total synthesis of ajoene, a major biologically active constituent of garlic. The instability of allicin as the only other known alternative starting material has led to the development of a reliable procedure for the synthesis of ajoene from simple building blocks that is also suitable for upscale operations.

The Pseudomonas aeruginosa Two-Component Regulator AlgR Directly Activates rsmA Expression in a Phosphorylation-Independent Manner.

Pseudomonas aeruginosa is an important pathogen of the immunocompromised, causing both acute and chronic infections. In cystic fibrosis (CF) patients, P. aeruginosa causes chronic disease. The impressive sensory network of P. aeruginosa allows the bacterium to sense and respond to a variety of stimuli found in diverse environments. Transcriptional regulators, including alternative sigma factors and response regulators, integrate signals changing gene expression, allowing P. aeruginosa to cause infection. The two-component transcriptional regulator AlgR is important in P. aeruginosa pathogenesis in both acute and chronic infections. In chronic infections, AlgR and the alternative sigma factor AlgU activate the genes responsible for alginate production. Previous work demonstrated that AlgU controls rsmA expression. RsmA is a posttranscriptional regulator that is antagonized by two small RNAs, RsmY and RsmZ. In this work, we demonstrate that AlgR directly activates rsmA expression from the same promoter as AlgU. In addition, phosphorylation was not necessary for AlgR activation of rsmA using algR and algZ mutant strains. AlgU and AlgR appear to affect the antagonizing small RNAs rsmY and rsmZ indirectly. RsmA was active in a mucA22 mutant strain using leader fusions of two RsmA targets, tssA1 and hcnA AlgU and AlgR were necessary for posttranscriptional regulation of tssA1 and hcnA Altogether, our work demonstrates that the alginate regulators AlgU and AlgR are important in the control of the RsmA posttranscriptional regulatory system. These findings suggest that RsmA plays an unknown role in mucoid strains due to AlgU and AlgR activities.IMPORTANCE P. aeruginosa infections are difficult to treat and frequently cause significant mortality in CF patients. Understanding the mechanisms of persistence is important. Our work has demonstrated that the alginate regulatory system also significantly impacts the posttranscriptional regulator system RsmA/Y/Z. We demonstrate that AlgR directly activates rsmA expression, and this impacts the RsmA regulon. This leads to the possibility that the RsmA/Y/Z system plays a role in helping P. aeruginosa persist during chronic infection. In addition, this furthers our understanding of the reach of the alginate regulators AlgU and AlgR.

Myeloid Cell-Specific Knockout of NFI-A Improves Sepsis Survival.

Myeloid progenitor-derived suppressor cells (MDSCs) arise from myeloid progenitors and suppress both innate and adaptive immunity. MDSCs expand during the later phases of sepsis in mice, promote immunosuppression, and reduce survival. Here, we report that the myeloid differentiation-related transcription factor nuclear factor I-A (NFI-A) controls MDSC expansion during sepsis and impacts survival. Unlike MDSCs, myeloid cells with conditional deletion of the Nfia gene normally differentiated into effector cells during sepsis, cleared infecting bacteria, and did not express immunosuppressive mediators. In contrast, ectopic expression of NFI-A in myeloid progenitors from NFI-A myeloid cell-deficient mice impeded myeloid cell maturation and promoted immune repressor function. Importantly, surviving septic mice with conditionally deficient NFI-A myeloid cells were able to respond to challenge with bacterial endotoxin by mounting an acute inflammatory response. Together, these results support the concept of NFI-A as a master molecular transcriptome switch that controls myeloid cell differentiation and maturation and that malfunction of this switch during sepsis promotes MDSC expansion that adversely impacts sepsis outcome.

Flat Drops, Elastic Sheets, and Microcapsules by Interfacial Assembly of a Bacterial Biofilm Protein, BslA.

Protein adsorption and assembly at interfaces provide a potentially versatile route to create useful constructs for fluid compartmentalization. In this context, we consider the interfacial assembly of a bacterial biofilm protein, BslA, at air-water and oil-water interfaces. Densely packed, high modulus monolayers form at air-water interfaces, leading to the formation of flattened sessile water drops. BslA forms elastic sheets at oil-water interfaces, leading to the production of stable monodisperse oil-in-water microcapsules. By contrast, water-in-oil microcapsules are unstable but display arrested rather than full coalescence on contact. The disparity in stability likely originates from a low areal density of BslA hydrophobic caps on the exterior surface of water-in-oil microcapsules, relative to the inverse case. In direct analogy with small molecule surfactants, the lack of stability of individual water-in-oil microcapsules is consistent with the large value of the hydrophilic-lipophilic balance (HLB number) calculated based on the BslA crystal structure. The occurrence of arrested coalescence indicates that the surface activity of BslA is similar to that of colloidal particles that produce Pickering emulsions, with the stability of partially coalesced structures ensured by interfacial jamming. Micropipette aspiration and flow in tapered capillaries experiments reveal intriguing reversible and nonreversible modes of mechanical deformation, respectively. The mechanical robustness of the microcapsules and the ability to engineer their shape and to design highly specific binding responses through protein engineering suggest that these microcapsules may be useful for biomedical applications.

What are the support experiences and needs of patients who have received bariatric surgery?

OBJECTIVE: To explore the support needs and experiences of patients who had received publicly or privately funded bariatric surgery and the importance of this support in mediating outcomes of surgery. METHODS: Seven semi-structured focus groups were conducted. A broad interview schedule guided the discussions which were audio-recorded and transcribed verbatim. Data were analysed thematically. RESULTS: Twenty-six women and 15 men with a mean age of 54 years (range 24-72) participated in the study. Participants described support needs from health professionals, significant others (family and friends), peers (bariatric surgery recipients) and the general community. Peer, dietetic and psychological support were identified as important factors influencing the outcomes (e.g. weight reduction or health improvement) or experience of bariatric surgery but were identified as infrequently received or inadequately provided. Psychological support was proposed as one of the most significant but commonly overlooked components of care. Support needs appeared higher in the first year post-surgery, when subsequent related or unrelated surgeries were required and following significant life change such as worsening health. For some participants, deficits in support appeared to negatively influence the experience or outcomes of surgery. CONCLUSION: Providers of bariatric surgery should discuss support needs and accessibility regularly with patients especially in the first year post-surgery and following significant change in a patient's life (e.g. declined health or childbirth). Nutrition, psychological and peer support (e.g. through support groups) may be especially important for some patients.

A systematic review of experiences of advanced practice nursing in general practice.

BACKGROUND: Despite efforts to achieve conceptual clarity, advanced practice nursing continues to reside in a liminal space, unable to secure ongoing recognition as a viable means of healthcare delivery. This is particularly evident in general practice where advanced practice role development is more fluid and generally less supported by the hierarchical structures evident in the hospital system. This review synthesises published qualitative studies reporting experiences of advanced practice nursing in general practice. The panoramic view provided by patients, nurses and doctors within this novel context, offers a fresh perspective on why advanced practice nurses have struggled to gain acceptance within the healthcare milieu. METHODS: We conducted a systematic review of qualitative studies that explored the experiences of patients, nurses and doctors who had contact with advanced practice nurses working in general practice. Published work from 1990 to June 2016 was located using CINAHL and PubMed. The full text of relevant studies was retrieved after reading the title and abstract. Critical appraisal was undertaken and the findings of included studies were analysed using the constant comparative method. Emergent codes were collapsed into sub-themes and themes. RESULTS: Twenty articles reporting the experiences of 486 participants were included. We identified one major theme: legitimacy; and three sub-themes: (1) establishing and maintaining confidence in the advanced practice nurse, (2) strengthening and weakening boundaries between general practitioners and advanced practice nurses and (3) establishing and maintaining the value of advanced practice nursing. CONCLUSIONS: We set out to describe experiences of advanced practice nursing in general practice. We discovered that general practitioners and patients continue to have concerns around responsibility, trust and accountability. Additionally, advanced practice nurses struggle to negotiate and clarify scopes of practice while general practitioners have trouble justifying the costs associated with advanced practice nursing roles. Therefore, much work remains to establish and maintain the legitimacy of advanced practice nursing in general practice.

Protein-Based Hydrogels for Tissue Engineering.

The tunable mechanical and structural properties of protein-based hydrogels make them excellent scaffolds for tissue engineering and repair. Moreover, using protein-based components provides the option to insert sequences associated with promoting both cellular adhesion to the substrate and overall cell growth. Protein-based hydrogel components are appealing for their structural designability, specific biological functionality, and stimuli-responsiveness. Here we present highlights in the field of protein-based hydrogels for tissue engineering applications including design requirements, components, and gel types.

Protein design: Past, present, and future.

Building on the pioneering work of Ho and DeGrado (J Am Chem Soc 1987, 109, 6751-6758) in the late 1980s, protein design approaches have revealed many fundamental features of protein structure and stability. We are now in the era that the early work presaged - the design of new proteins with practical applications and uses. Here we briefly survey some past milestones in protein design, in addition to highlighting recent progress and future aspirations.

Novel insights into protein misfolding diseases revealed by ion mobility-mass spectrometry.

Amyloid disorders incorporate a wide range of human diseases arising from the failure of a specific peptide or protein to adopt, or remain in, its native functional conformational state. These pathological conditions, such as Parkinson's disease, Alzheimer's disease and Huntington's disease are highly debilitating, exact enormous costs on both individuals and society, and are predicted to increase in prevalence. Consequently, they form the focus of a topical and rich area of current scientific research. A major goal in attempts to understand and treat protein misfolding diseases is to define the structures and interactions of protein species intermediate between fully folded and aggregated, and extract a description of the aggregation process. This has proven a difficult task due to the inability of traditional structural biology approaches to analyze structurally heterogeneous systems. Continued developments in instrumentation and analytical approaches have seen ion mobility-mass spectrometry (IM-MS) emerge as a complementary approach for protein structure determination, and in some cases, a structural biology tool in its own right. IM-MS is well suited to the study of protein misfolding, and has already yielded significant structural information for selected amyloidogenic systems during the aggregation process. This review describes IM-MS for protein structure investigation, and provides a summary of current research highlighting how this methodology has unequivocally and unprecedentedly provided structural and mechanistic detail pertaining to the oligomerization of a variety of disease related proteins.