Antiganglioside antibody frequency in routine clinical care settings.

BACKGROUND AND PURPOSE: Antiganglioside antibodies (AGAs) might be involved in the etiopathogenesis of many neurological diseases, such as Miller-Fisher syndrome (MFS) and Guillain-Barré syndrome (GBS). Available comprehensive reference data regarding AGA positivity rates and cross-responsiveness among AGAs (where one line immunoblot is positive for ≥1 AGA) during routine clinical care are scant. METHODS: In this 10-year monocentric retrospective study, 3560 immunoglobulin (Ig) G and IgM line blots (GA Generic Assays' Anti-Ganglioside Dot kit) obtained using cerebrospinal fluid (CSF) and serum samples from 1342 patients were analyzed for AGA positivity in terms of 14 diagnosis categories and AGA cross-responsiveness. RESULTS: Of all 3560 line blots 158 (4.4%) and of all CSF samples 0.4% (4/924) CSF line blots were AGA positive. For serum IgG, blots with positivity rates higher than the standard deviation of 15.6% were associated with MFS (GD3, GD1a, GT1a and GQ1b) and acute motor axonal neuropathy (AMAN) (GM1, GD1a and GT1a). For serum IgM, blots with positivity rates higher than the standard deviation of 8.1% were associated with AMAN (GM2, GT1a and GQ1b), MFS (GM1, GT1a and GQ1b), multifocal motor neuropathy (MMN) (GM1, GM2 and GQ1b) and chronic inflammatory demyelinating polyneuropathy (CIDP) (GM1). Cross-responsiveness was observed in 39.6% of all positive serum AGA. CONCLUSIONS: Testing for AGAs during routine clinical care rarely led to positive findings, both in serum and even less in CSF, except for the diagnoses AMAN, MFS, MMN and CIDP. Nonspecific findings found as cross-responsiveness between different AGA samples occur frequently, impacting the positivity of most AGA subtypes.

Continuous and Non-Invasive Lactate Monitoring Techniques in Critical Care Patients.

Lactate, once merely regarded as an indicator of tissue hypoxia and muscular fatigue, has now gained prominence as a pivotal biomarker across various medical disciplines. Recent research has unveiled its critical role as a high-value prognostic marker in critical care medicine. The current practice of lactate detection involves periodic blood sampling. This approach is invasive and confined to measurements at six-hour intervals, leading to resource expenditure, time consumption, and patient discomfort. This review addresses non-invasive sensors that enable continuous monitoring of lactate in critical care patients. After the introduction, it discusses the iontophoresis system, followed by a description of the structural materials that are universally employed to create an interface between the integumentary system and the sensor. Subsequently, each method is detailed according to its physical principle, outlining its advantages, limitations, and pertinent aspects. The study concludes with a discussion and conclusions, aiming at the design of an intelligent sensor (Internet of Medical Things or IoMT) to facilitate continuous lactate monitoring and enhance the clinical decision-making support system in critical care medicine.

Rapid binding to protofilament edge sites facilitates tip tracking of EB1 at growing microtubule plus-ends.

EB1 is a key cellular protein that delivers regulatory molecules throughout the cell via the tip-tracking of growing microtubule plus-ends. Thus, it is important to understand the mechanism for how EB1 efficiently tracks growing microtubule plus-ends. It is widely accepted that EB1 binds with higher affinity to GTP-tubulin subunits at the growing microtubule tip, relative to GDP-tubulin along the microtubule length. However, it is unclear whether this difference in affinity alone is sufficient to explain the tip-tracking of EB1 at growing microtubule tips. Previously, we found that EB1 binds to exposed microtubule protofilament-edge sites at a ~70 fold faster rate than to closed-lattice sites, due to diffusional steric hindrance to binding. Thus, we asked whether rapid protofilament-edge binding could contribute to efficient EB1 tip tracking. A computational simulation with differential EB1 on-rates based on closed-lattice or protofilament-edge binding, and with EB1 off-rates that were dependent on the tubulin hydrolysis state, robustly recapitulated experimental EB1 tip tracking. To test this model, we used cell-free biophysical assays, as well as live-cell imaging, in combination with a Designed Ankyrin Repeat Protein (DARPin) that binds exclusively to protofilament-edge sites, and whose binding site partially overlaps with the EB1 binding site. We found that DARPin blocked EB1 protofilament-edge binding, which led to a decrease in EB1 tip tracking on dynamic microtubules. We conclude that rapid EB1 binding to microtubule protofilament-edge sites contributes to robust EB1 tip tracking at the growing microtubule plus-end.

Systematic Review of Automated Diuresis Measurement in Critically Ill Patients.

The measurement of urinary flow is a vital medical indicator for critically ill patients in intensive care units. However, there is a clinical need to automate the real-time measurement of diuresis using Internet of Medical Things devices, allowing continuous monitoring of urine flow. A systematic review of scientific literature, patents, and available commercial products was conducted, leading to the conclusion that there is no suitable device to fulfill this need. We identified six characteristics that such a device should possess: minimizing contact with urine, detecting changes in flow patterns, the ability to record minute-by-minute data, capable of sending early alerts, not relying on exclusive disposable components, and being user-friendly for clinical professionals. Additionally, cost-effectiveness is crucial, encompassing the device, infrastructure, maintenance, and usage.

Development of a urinometer for automatic measurement of urine flow in catheterized patients.

Urinary flow measurement and colorimetry are vital medical indicators for critically ill patients in intensive care units. However, there is a clinical need for low-cost, continuous urinary flow monitoring devices that can automatically and in real-time measure urine flow. This need led to the development of a non-invasive device that is easy to use and does not require proprietary disposables. The device operates by detecting urine flow using an infrared barrier that returns an unequivocal pattern, and it is capable of measuring the volume of liquid in real-time, storing the history with a precise date, and returning alarms to detect critical trends. The device also has the ability to detect the color of urine, allowing for extended data and detecting problems in catheterized patients such as hematuria. The device is proposed as an automated clinical decision support system that utilizes the concept of the Internet of Medical Things. It works by using a LoRa communication method with the LoRaWAN protocol to maximize the distance to access points, reducing infrastructure costs in massive deployments. The device can send data wirelessly for remote monitoring and allows for the collection of data on a dashboard in a pseudonymous way. Tests conducted on the device using a gold standard medical grade infusion pump and fluid densities within the 1.005 g/ml to 1.030 g/ml urine density range showed that droplets were satisfactorily captured in the range of flows from less than 1 ml/h to 500 ml/h, which are acceptable ranges for urinary flow. Errors ranged below 15%, when compared to the values obtained by the hospital infusion pump used as gold standard. Such values are clinically adequate to detect changes in diuresis patterns, specially at low urine output ranges, related to renal disfunction. Additionally, tests carried out with different color patterns indicate that it detects different colors of urine with a precision in detecting RGB values <5%. In conclusion, the results suggest that the device can be useful in automatically monitoring diuresis and colorimetry in real-time, which can facilitate the work of nursing and provide automatic decision-making support to intensive care physicians.

Hantavirus in Panama: Twenty Years of Epidemiological Surveillance Experience.

Twenty years have passed since the emergence of hantavirus zoonosis in Panama at the beginning of this millennium. We provide an overview of epidemiological surveillance of hantavirus disease (hantavirus pulmonary syndrome and hantavirus fever) during the period 1999-2019 by including all reported and confirmed cases according to the case definition established by the health authority. Our findings reveal that hantavirus disease is a low-frequency disease, affecting primarily young people, with a relatively low case-fatality rate compared to other hantaviruses in the Americas (e.g., ANDV and SNV). It presents an annual variation with peaks every 4-5 years and an interannual variation influenced by agricultural activities. Hantavirus disease is endemic in about 27% of Panama, which corresponds to agroecological conditions that favor the population dynamics of the rodent host, Oligoryzomys costaricensis and the virus (Choclo orthohantavirus) responsible for hantavirus disease. However, this does not rule out the existence of other endemic areas to be characterized. Undoubtedly, decentralization of the laboratory test and dissemination of evidence-based surveillance guidelines and regulations have standardized and improved diagnosis, notification at the level of the primary care system, and management in intensive care units nationwide.

Electroencefalografía básica para monitores de profundidad anestésica / Basic electroencephalography for anesthesia depth monitors

Resumen: Los monitores de profundidad anestésica permiten guiar el estado hipnótico del paciente durante la anestesia general. Debido a su sencillez, tradicionalmente se han empleado índices de profundidad anestésica, obtenidos a través del procesamiento del electroencefalograma mediante algoritmos matemáticos, para orientar la monitorización del nivel de consciencia. Sus beneficios han sido ampliamente recogidos en la literatura científica; sin embargo, no están exentos de importantes limitaciones. No todos los anestésicos actúan en las mismas dianas moleculares ni dichos índices tienen en cuenta las características propias del paciente (comorbilidades, edades extremas, etcétera). Estas limitaciones podrían reducirse si interpretamos directamente toda la información que nos ofrecen los monitores. Presentamos una revisión que describe los conceptos básicos necesarios para su valoración directa, así como su correlación con los estados de profundidad anestésica del paciente.

Abstract: Anesthesia depth monitors allow to guide the patient's hypnotic state during general anesthesia. Traditionally, anesthetic depth indices have been used due to their simplicity to guide the monitoring of the level of consciousness. They have been obtained by processing the electroencephalogram using mathematical algorithms and their benefits have been widely reported in the scientific literature. However, they are not exempt from important limitations. Neither all anesthetics act on the same molecular targets, nor these mentioned indices take into account the patient's own characteristics (comorbidities, extreme ages, etc.). These limitations could be far reduced if we are able to understand all the information provided by the monitors. We present a review describing the basic concepts necessary for its direct assessment, as well as their correlation with the patient's anesthetic depth states.

Robust microtubule dynamics facilitate low-tension kinetochore detachment in metaphase.

During mitosis, sister chromatids are stretched apart at their centromeres via their attachment to oppositely oriented kinetochore microtubules. This stretching generates inwardly directed tension across the separated sister centromeres. The cell leverages this tension signal to detect and then correct potential errors in chromosome segregation, via a mechanical tension signaling pathway that detaches improperly attached kinetochores from their microtubules. However, the sequence of events leading up to these detachment events remains unknown. In this study, we used microfluidics to sustain and observe low-tension budding yeast metaphase spindles over multiple hours, allowing us to elucidate the tension history prior to a detachment event. We found that, under conditions in which kinetochore phosphorylation weakens low-tension kinetochore-microtubule connections, the mechanical forces produced via the dynamic growth and shortening of microtubules is required to efficiently facilitate detachment events. Our findings underscore the critical role of robust kinetochore microtubule dynamics in ensuring the fidelity of chromosome segregation during mitosis.

Influence of the Results of Control of Intakes, Proteins and Anthropometry Nutritional Screening, Sarcopenia and Body Composition on the Clinical Evolution of Hospitalized Patients.

(1) Background: Hospital malnutrition and sarcopenia are common in inpatients and are associated with worse prognosis. Our objective is to determine the association of the positivity of CIPA (Control of Intakes, Proteins and Anthropometry) nutrition screening tool and sarcopenia and evaluate its prognostic implications (length of stay, readmissions and mortality) as well as different components of body composition. (2) Methodology: Cross-sectional single-center study and prospective six months follow-up for prognostic variables. On admission, CIPA and EWGSOP2 criteria were assessed. (3) Results: Four hundred inpatients, a median of 65.71 years old and 83.6% with high comorbidity, were evaluated. In total, 34.8% had positive CIPA and 19.3% sarcopenia. Positive CIPA and sarcopenia had worse results in body composition (fat mass (FM), fat-free mass (FFM) and appendicular skeletal muscle mass index (ASMI)) and dynamometry. Positive CIPA is significantly associated with worse prognosis (mortality (OR = 1.99), readmissions (OR = 1.86) and length of stay (B = 0.19)). Positive CIPA and sarcopenia combined are associated with a tendency to higher mortality (OR = 2.1, p = 0.088). Low hand grip strength (HGS) is significantly related to a higher length of stay (B = -0.12). (4) Conclusions: In hospitalized patients, malnutrition independently and combined with sarcopenia is associated with a worse prognosis but not body composition. Low HGS is related to a higher length of stay.

Improved Stability of Human CGI-58 Induced by Phosphomimetic S237E Mutation.

In lipolysis, the activating function of CGI-58 is regulated by its interaction with perilipin 1 (PLIN1) localized on the lipid droplet (LD), and its release is controlled by phosphorylation. Once lipolysis is stimulated by catecholamines, protein kinase A (PKA)-mediated phosphorylation enables the dissociation of the CGI-58/PLIN1 complex, thereby recruiting adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) to initiate fatty acid release. It has been shown that mouse CGI-58 mutant S239E, which mimics the phosphorylation of this residue, is able to dissociate from the CGI-58/PLIN1 complex and activate ATGL. Here, we analyze the stabilizing effect on human CGI-58 of a triple tryptophan to alanine mutant (3WA) on the LD-binding motif, as well as a quadruple mutant in which the phosphomimetic S237E substitution was introduced to the 3WA construct (3WA/S237E). We found that tryptophan residues promote wild-type (WT) protein aggregation in solution since their substitution for alanine residues favors the presence of the monomer. Our experimental data showed increased thermal stability and solubility of 3WA/S237E protein compared to the 3WA mutant. Moreover, the 3WA/S237E protein showed proper folding and a functional binding site for oleoyl-CoA. The analysis of a bioinformatic three-dimensional (3D) model suggests an intramolecular interaction between the phosphomimetic glutamic acid and a residue of the α/ß hydrolase core. This could explain the increased solubility and stability observed in the 3WA/S237E mutant and evidences the possible role of serine 237 phosphorylation.

Kinesin-14 motors participate in a force balance at microtubule plus-ends to regulate dynamic instability.

Kinesin-14 molecular motors represent an essential class of proteins that bind microtubules and walk toward their minus-ends. Previous studies have described important roles for Kinesin-14 motors at microtubule minus-ends, but their role in regulating plus-end dynamics remains controversial. Kinesin-14 motors have been shown to bind the EB family of microtubule plus-end binding proteins, suggesting that these minus-end-directed motors could interact with growing microtubule plus-ends. In this work, we explored the role of minus-end-directed Kinesin-14 motor forces in controlling plus-end microtubule dynamics. In cells, a Kinesin-14 mutant with reduced affinity to EB proteins led to increased microtubule lengths. Cell-free biophysical microscopy assays were performed using Kinesin-14 motors and an EB family marker of growing microtubule plus-ends, Mal3, which revealed that when Kinesin-14 motors bound to Mal3 at growing microtubule plus-ends, the motors subsequently walked toward the minus-end, and Mal3 was pulled away from the growing microtubule tip. Strikingly, these interactions resulted in an approximately twofold decrease in the expected postinteraction microtubule lifetime. Furthermore, generic minus-end-directed tension forces, generated by tethering growing plus-ends to the coverslip using λ-DNA, led to an approximately sevenfold decrease in the expected postinteraction microtubule growth length. In contrast, the inhibition of Kinesin-14 minus-end-directed motility led to extended tip interactions and to an increase in the expected postinteraction microtubule lifetime, indicating that plus-ends were stabilized by nonmotile Kinesin-14 motors. Together, we find that Kinesin-14 motors participate in a force balance at microtubule plus-ends to regulate microtubule lengths in cells.

Oxidative stress pathogenically remodels the cardiac myocyte cytoskeleton via structural alterations to the microtubule lattice.

In the failing heart, the cardiac myocyte microtubule network is remodeled, which contributes to cellular contractile failure and patient death. However, the origins of this deleterious cytoskeletal reorganization are unknown. We now find that oxidative stress, a condition characteristic of heart failure, leads to cysteine oxidation of microtubules. Our electron and fluorescence microscopy experiments revealed regions of structural damage within the microtubule lattice that occurred at locations of oxidized tubulin. The incorporation of GTP-tubulin into these damaged, oxidized regions led to stabilized "hot spots" within the microtubule lattice, which suppressed the shortening of dynamic microtubules. Thus, oxidative stress may act inside of cardiac myocytes to facilitate a pathogenic shift from a sparse microtubule network into a dense, aligned network. Our results demonstrate how a disease condition characterized by oxidative stress can trigger a molecular oxidation event, which likely contributes to a toxic cellular-scale transformation of the cardiac myocyte microtubule network.

Purification and characterization of an FeII- and α-ketoglutarate-dependent xanthine hydroxylase from Aspergillus oryzae.

XanA is an FeII- and α-ketoglutarate-dependent enzyme responsible for the conversion of xanthine to uric acid. It is unique to fungi and it was first described in Aspergillus nidulans. In this work, we present the preliminary characterization of the XanA enzyme from Aspergillus oryzae, a relevant fungus in food production in Japan. The XanA protein (GenBank BAE56701.1) was expressed as a recombinant protein in Escherichia coli BL21 (DE3) Arctic cells. Initial purification assays showed low protein solubility; therefore, the buffer composition was optimized using a fluorescence-based thermal shift assay. The protein was stabilized in solution in the presence of either 600 µM xanthine, 1 M NaCl, 600 µM α-ketoglutarate or 20% glycerol, which increases the melting temperature (Tm) by 2, 4, 5 and 6 °C respectively. The XanA protein was purified by following a three-step purification protocol. The nickel affinity purified protein was subjected to ion-exchange chromatography once the N-terminal 6XHis-tag had been successfully removed, followed by size-exclusion purification. Dynamic light scattering experiments showed that the purified protein was monodisperse and behaved as a monomer in solution. Preliminary activity assays in the presence of xanthine, α-ketoglutarate, and iron suggest that the enzyme is an iron- and α-ketoglutarate-dependent xanthine dioxygenase. Furthermore, the enzyme's optimum activity conditions were determined to be 25 °C, pH of 7.2, HEPES buffer, and 1% of glycerol. In conclusion, we established the conditions to purify the XanA enzyme from A. oryzae in its active form from E. coli bacteria and determined the optimal activity conditions.

Five-year outcome of peripherally inserted central catheters in adults: a separated infectious and thrombotic complications analysis.

OBJECTIVE: To assess infectious and thrombotic complications of peripherally inserted central catheters (PICCs) in adults. DESIGN: A 5-year prospective cohort study. SETTING: Tertiary-care teaching hospital in Seville, Spain. PATIENTS: Adult patients undergoing PICC insertion. METHODS: Catheter-associated bloodstream infection (CABSI) including catheter-related bloodstream infection (CRBSI), primary bacteremia (PB), and upper extremity deep vein thrombosis (UEDVT) were recorded. Independent predictors of complications were assessed by multivariate analysis. RESULTS: In total, 1,142 PICCs were inserted, with 153,191 catheter days (median, 79). Complications included 66 cases of CABSI (5.78%; 0.43‰ catheter days), 38 cases of CRBSI (3.33%; 0.25‰ catheter days), 28 cases of PB (2.45%; 0.18‰ catheter days), and 23 cases of UEDVT (2.01%; 0.15‰ catheter days). The median times to infection were 24, 41, and 60 days for CRBSI, PB, and UEDVT, respectively. Parenteral nutrition (odds ratio [OR], 3.40; 95% confidence interval [CI], 1.77-6.52) and admission to the hematology ward (OR, 4.90; 95% CI, 2.25-10.71) were independently associated with CRBSI and PB, respectively. Admission to the hematology ward (OR, 12.46; 95% CI, 2.49-62.50) or to the oncology ward (OR, 7.89; 95% CI, 1.77-35.16) was independently associated with UEDVT. The crude mortality rate was 24.8%. Only 2 patients died of complications. CONCLUSIONS: PICCs showed a low rate of thrombotic and infectious complications. Compared to PB, CRBSI showed significantly different risk factors, a higher incidence density per catheter days, and a shorter median time to infection. Separate analyses of CRBSI and PB are more specific and clinically useful when analyzing infectious complications.

The RNA Polymerase α Subunit Recognizes the DNA Shape of the Upstream Promoter Element.

We demonstrate here that the α subunit C-terminal domain of Escherichia coli RNA polymerase (αCTD) recognizes the upstream promoter (UP) DNA element via its characteristic minor groove shape and electrostatic potential. In two compositionally distinct crystallized assemblies, a pair of αCTD subunits bind in tandem to the UP element consensus A-tract that is 6 bp in length (A6-tract), each with their arginine 265 guanidinium group inserted into the minor groove. The A6-tract minor groove is significantly narrowed in these crystal structures, as well as in computationally predicted structures of free and bound DNA duplexes derived by Monte Carlo and molecular dynamics simulations, respectively. The negative electrostatic potential of free A6-tract DNA is substantially enhanced compared to that of generic DNA. Shortening the A-tract by 1 bp is shown to "knock out" binding of the second αCTD through widening of the minor groove. Furthermore, in computationally derived structures with arginine 265 mutated to alanine in either αCTD, either with or without the "knockout" DNA mutation, contact with the DNA is perturbed, highlighting the importance of arginine 265 in achieving αCTD-DNA binding. These results demonstrate that the importance of the DNA shape in sequence-dependent recognition of DNA by RNA polymerase is comparable to that of certain transcription factors.

Bioinformatic Analysis and Biophysical Characterization Reveal Structural Disorder in G0S2 Protein.

G0S2 is a small protein of 103 residues in length that is involved in multiple cellular processes. To date, several reports have shown that G0S2 functions by making direct protein-protein interactions with key proteins. In lipolysis, G0S2 specifically interacts with adipose triglyceride lipase, inhibiting its activity and resulting in lipolysis being downregulated. In a similar way, G0S2 also participates in the regulation of apoptosis, cell proliferation, and oxidative phosphorylation; however, information regarding G0S2 structural and biophysical properties is limited. In this work, we conducted a comparative structural analysis of human and mouse G0S2 proteins. Bioinformatics suggests the presence of a disordered C-terminal region in human G0S2. Experimental characterization by size-exclusion chromatography and dynamic light scattering showed that human and mouse G0S2 have different hydrodynamic properties. In comparison to the mouse G0S2, which behaves similar to a globular protein, the human G0S2 shows an elongated conformation, most likely by displaying a disordered C-terminal region. Further analysis of hydrodynamic properties under denaturing conditions suggests the presence of a structural element in the mouse protein that undergoes an order to disorder transition at low urea concentration. Structural analysis by circular dichroism revealed that in native conditions, both proteins are mainly unstructured, showing the presence of beta sheet structures. Further analysis of CD data suggests that both proteins belong to the premolten globule family of intrinsically disordered proteins. We suggest that the intrinsic disorder observed in the G0S2 protein may facilitate its interaction with multiple partners in the regulation of cellular metabolism.

A Novel Use of Aortic Stent Graft Components in Massive Venous Retroperitoneal Hematoma.

Venous retroperitoneal hematoma (VRH) can present as a sudden-onset life-threatening condition. Unlike arterial hematoma, VRH is difficult to treat because of anatomic and structural considerations. Moreover, because VRH is rare and iatrogenic, there are no commercially available devices to treat this problem. We present a novel use of aortic stent graft components to treat a large, life-threatening VRH.

Bilateral compensatory postural adjustments to a unilateral perturbation in subjects with chronic ankle instability.

BACKGROUND: To evaluate the magnitude of bilateral compensatory postural adjustments in response to a unilateral sudden inversion perturbation in subjects with chronic ankle instability. METHODS: 24 athletes with chronic ankle instability (14 with functional ankle instability, 10 with mechanical ankle instability) and twenty controls participated in this study. The bilateral electromyography of ankle muscles was collected during a unilateral sudden ankle inversion to assess the magnitude of subcortical and voluntary compensatory postural adjustments in both the perturbed and the contralateral limb (support limb). FINDINGS: In the support position, compared to the control group, the group with functional ankle instability presented decreased compensatory postural adjustments of the tibialis anterior in both the injured and the uninjured limbs in the support position and of the soleus in the uninjured limb. In the side of the perturbation, participants with functional ankle instability presented decreased soleus compensatory postural adjustments in the uninjured limb when compared to the control group. Increased values of soleus and peroneal brevis compensatory postural adjustments were observed in the group with mechanical instability when compared to the control group and to the group with functional ankle instability. INTERPRETATION: Subjects with functional ankle instability present bilateral impairment of compensatory postural adjustments of the tibialis anterior in a support position and of the soleus of the uninjured limb regardless of the position. Subjects with mechanical instability present bilateral increase of these adjustments in the peroneal brevis regardless of the position and in the soleus muscle in the side of the perturbation.

A competent catalytic active site is necessary for substrate induced dimer assembly in triosephosphate isomerase.

The protozoan parasite Trichomonas vaginalis contains two nearly identical triosephosphate isomerases (TvTIMs) that dissociate into stable monomers and dimerize upon substrate binding. Herein, we compare the role of the "ball and socket" and loop 3 interactions in substrate assisted dimer assembly in both TvTIMs. We found that point mutants at the "ball" are only 39 and 29-fold less catalytically active than their corresponding wild-type counterparts, whereas Δloop 3 deletions are 1502 and 9400-fold less active. Point and deletion mutants dissociate into stable monomers. However, point mutants assemble as catalytic competent dimers upon binding of the transition state substrate analog PGH, whereas loop 3 deletions remain monomeric. A comparison between crystal structures of point and loop 3 deletion monomeric mutants illustrates that the catalytic residues in point mutants and wild-type TvTIMs are maintained in the same orientation, whereas the catalytic residues in deletion mutants show an increase in thermal mobility and present structural disorder that may hamper their catalytic role. The high enzymatic activity present in monomeric point mutants correlates with the formation of dimeric TvTIMs upon substrate binding. In contrast, the low activity and lack of dimer assembly in deletion mutants suggests a role of loop 3 in promoting the formation of the active site as well as dimer assembly. Our results suggest that in TvTIMs the active site is assembled during dimerization and that the integrity of loop 3 and ball and socket residues is crucial to stabilize the dimer.