Studying the heterogeneous pathogenesis of canine diabetes: Observational characterization of an island population.

BACKGROUND: Canine diabetes mellitus has mostly been studied in northern European, Australian and American populations, whereas other regions have received less attention. OBJECTIVES: We evaluated the epidemiological, clinical and histopathological features of diabetic dogs in Gran Canaria, Spain. METHODS: Prevalence and incidence were estimated. Clinical features were analysed, and serum and genomic DNA were obtained. Dogs with presumed idiopathic or immune-mediated diabetes, were DLA-typed and antibodies against GAD65 and IA-2 were assessed. Pancreases from ten diabetic dogs were examined and compared with pancreases from non-diabetic dogs. RESULTS AND CONCLUSIONS: Twenty-nine diabetic dogs were identified in a population of 5,213 (prevalence: 0.56%; incidence: 0.37%). Most were female (79%) and sexually intact (87% of females, 83% of males). Diabetes secondary to dioestrus (55.2%) and insulin-deficient diabetes (20.7%) were the most frequent types. Antibodies against GAD65 and IA-2 were identified in two out of five cases and DLA-genotyping revealed novel haplotypes. Breed distribution differed between diabetic and non-diabetic dogs. Reduced number of pancreatic islets and ß-cell mass were observed, with vacuolation of islet cells and ductal epithelium. In this population, where neutering is not standard practice, diabetes secondary to dioestrus is the most frequent diabetes subtype. Genetic susceptibility also differed from previous studies. These results support the heterogeneous pathogenesis of canine diabetes.

Acute Effects of a Polyphenol-Rich Leaf Extract of Mangifera indica L. (Zynamite) on Cognitive Function in Healthy Adults: A Double-Blind, Placebo-Controlled Crossover Study.

Extracts made from the leaves of the mango food plant (Mangifera indica L., Anacardiaceae) have a long history of medicinal usage, most likely due to particularly high levels of the polyphenol mangiferin. In rodent models, oral mangiferin protects cognitive function and brain tissue from a number of challenges and modulates cerebro-electrical activity. Recent evidence has confirmed the latter effect in healthy humans following a mangiferin-rich mango leaf extract using quantitative electroencephalography (EEG). The current study therefore investigated the effects of a single dose of mango leaf extract, standardised to contain >60% mangiferin (Zynamite®), on cognitive function and mood. This study adopted a double-blind, placebo-controlled cross-over design in which 70 healthy young adults (18 to 45 years) received 300 mg mango leaf extract and a matched placebo, on separate occasions, separated by at least 7 days. On each occasion, cognitive/mood assessments were undertaken pre-dose and at 30 min, 3 h and 5 h post-dose using the Computerised Mental Performance Assessment System (COMPASS) assessment battery and the Profile of Mood States (POMS). The results showed that a single dose of 300 mg mango leaf extract significantly improved performance accuracy across the tasks in the battery, with domain-specific effects seen in terms of enhanced performance on an 'Accuracy of Attention' factor and an 'Episodic Memory' factor. Performance was also improved across all three tasks (Rapid Visual Information Processing, Serial 3s and Serial 7s subtraction tasks) that make up the Cognitive Demand Battery sub-section of the assessment. All of these cognitive benefits were seen across the post-dose assessments (30 min, 3 h, 5 h). There were no interpretable treatment related effects on mood. These results provide the first demonstration of cognition enhancement following consumption of mango leaf extract and add to previous research showing that polyphenols and polyphenol rich extracts can improve brain function.

Central nervous system activities of extract Mangifera indica L.

ETHNOBOTANICAL RELEVANCE: Leaves of Mangifera indica L. have folk-uses in tropical regions of the world as health teas, as a remedy for exhaustion and fatigue, as a vegetable, and as a medicine. Mangifera indica leaf extract (MLE) had previously been demonstrated to alter brain electrical activity in-vivo. The aim of the present series of studies was to investigate whether mangiferin, a major compound in leaves and in MLE, is responsible for the neurocognitive activity of MLE, and if the CNS activities of MLE have translational potential. MATERIALS AND METHODS: MLE, tradename Zynamite, is produced by Nektium Pharma, Spain. Isolated mangiferin was tested in-vitro in radioligand binding and enzyme inhibition studies against 106 CNS targets. Changes in the electroencephalograms (EEG's) of MLE and mangiferin were recorded in-vivo from four brain regions. Two double blind randomized placebo-controlled crossover clinical trials were conducted, each with 16 subjects. At 90 min and at 60 min respectively, after oral intake of 500 mg MLE, EEG recordings, psychometric tests, mood state, and tolerability were studied. RESULTS: Isolated mangiferin is a selective inhibitor of catechol-O-methyltransferase (COMT) with an IC50 of 1.1 µM, with no activity on the CNS targets of caffeine. Both mangiferin and MLE induce similar changes in long-term potentiation (LTP) in the hippocampus in-vitro, and induce a similar pattern of EEG changes in-vivo. In both translational clinical trials MLE was well tolerated, with no cardiovascular side-effects. In both studies MLE caused significant spectral changes in brain electrical activity in cortical regions during cognitive challenges, different to the attenuated spectral changes induced by caffeine. There were no significant changes in the psychometric tests other than reaction time for all groups. In the second study there was a trend to faster reaction time within group for MLE (p = 0.066) and the percentage improvement in reaction time for MLE compared to placebo was significant (p = 0.049). In the first study MLE improved all scores for Profile of Mood States (POMS), with the score for "fatigue" significantly improved (p = 0.015); in the second study the POMS score for "dejection" was improved in the caffeine group, p = 0.05. CONCLUSIONS: Mangiferin is a COMT inhibitor of moderate potency and is the major CNS-active compound in MLE. Both mangiferin and MLE increase hippocampal LTP in-vitro, and induce a similar pattern of changes in brain electrical activity in-vivo. While the translational clinical trials of MLE are limited by being single dose studies in a small number of subjects, they provide the first clinical evidence that the extract is well tolerated with no cardiovascular side-effects, can induce changes in brain electrical activity, may give a faster reaction time, and decrease fatigue. These CNS activities support the reported folk-uses use of mango leaf tea as a substitute for tea and as a traditional remedy for fatigue and exhaustion. Extract Mangifera indica L., Zynamite, has nootropic potential, and larger clinical studies are needed to realise this potential.

Supplementation with a Mango Leaf Extract (Zynamite®) in Combination with Quercetin Attenuates Muscle Damage and Pain and Accelerates Recovery after Strenuous Damaging Exercise.

Prolonged or unusual exercise may cause exercise-induced muscle damage (EIMD). To test whether Zynamite®, a mango leaf extract rich in the natural polyphenol mangiferin, administered in combination with quercetin facilitates recovery after EIMD, 24 women and 33 men were randomly assigned to two treatment groups matched by sex and 5 km running performance, and ran a 10 km race followed by 100 drop jumps to elicit EIMD. One hour before the competition, and every 8 hours thereafter for 24 hours, they ingested placebo (728 mg of maltodextrin) or 140 mg of Zynamite® combined with 140 mg of quercetin (double-blind). Although competition times were similar, polyphenol supplementation attenuated the muscle pain felt after the competition (6.8 ± 1.5 and 5.7 ± 2.2 a.u., p = 0.035) and the loss of jumping performance (9.4 ± 11.5 and 3.9 ± 5.2%, p = 0.036; p = 0.034) and mechanical impulse (p = 0.038) 24 hours later. The polyphenols attenuated the increase of serum myoglobin and alanine aminotransferase in men, but not in women (interaction p < 0.05). In conclusion, a single dose of 140 mg Zynamite® combined with 140 mg of quercetin, administered one hour before competition, followed by three additional doses every eight hours, attenuates muscle pain and damage, and accelerates the recovery of muscle performance.

A Single Dose of The Mango Leaf Extract Zynamite® in Combination with Quercetin Enhances Peak Power Output During Repeated Sprint Exercise in Men and Women.

The mango leaf extract rich in mangiferin Zynamite® improves exercise performance when combined with luteolin or quercetin ingested at least 48 h prior to exercise. To determine whether a single dose of Zynamite® administered 1 h before exercise increases repeated-sprint performance, 20 men and 20 women who were physically active were randomly assigned to three treatments following a double-blind cross-over counterbalanced design. Treatment A, 140 mg of Zynamite®, 140 mg of quercetin, 147.7 mg of maltodextrin, and 420 mg of sunflower lecithin; Treatment B, 140 mg of Zynamite®, 140 mg of quercetin, and 2126 mg of maltodextrin and Treatment C, 2548 mg of maltodextrin (placebo). Subjects performed three Wingate tests interspaced by 4 min and a final 15 s sprint after ischemia. Treatments A and B improved peak power output during the first three Wingates by 2.8% and 3.8%, respectively (treatment x sprint interaction, p = 0.01). Vastus Lateralis oxygenation (NIRS) was reduced, indicating higher O2 extraction (treatment × sprint interaction, p = 0.01). Improved O2 extraction was observed in the sprints after ischemia (p = 0.008; placebo vs. mean of treatments A and B). Blood lactate concentration was 5.9% lower after the ingestion of Zynamite® with quercetin in men (treatment by sex interaction, p = 0.049). There was a higher Vastus Lateralis O2 extraction during 60 s ischemia with polyphenols (treatment effect, p = 0.03), due to the greater muscle VO2 in men (p = 0.001). In conclusion, a single dose of Zynamite® combined with quercetin one hour before exercise improves repeated-sprint performance and muscle O2 extraction and mitochondrial O2. consumption during ischemia. No advantage was obtained from the addition of phospholipids.

Enhancement of Exercise Performance by 48 Hours, and 15-Day Supplementation with Mangiferin and Luteolin in Men.

The natural polyphenols mangiferin and luteolin have free radical-scavenging properties, induce the antioxidant gene program and down-regulate the expression of superoxide-producing enzymes. However, the effects of these two polyphenols on exercise capacity remains mostly unknown. To determine whether a combination of luteolin (peanut husk extract containing 95% luteolin, PHE) and mangiferin (mango leave extract (MLE), Zynamite®) at low (PHE: 50 mg/day; and 140 mg/day of MLE containing 100 mg of mangiferin; L) and high doses (PHE: 100 mg/day; MLE: 420 mg/day; H) may enhance exercise performance, twelve physically active men performed incremental exercise to exhaustion, followed by sprint and endurance exercise after 48 h (acute effects) and 15 days of supplementation (prolonged effects) with polyphenols or placebo, following a double-blind crossover design. During sprint exercise, mangiferin + luteolin supplementation enhanced exercise performance, facilitated muscle oxygen extraction, and improved brain oxygenation, without increasing the VO2. Compared to placebo, mangiferin + luteolin increased muscle O2 extraction during post-exercise ischemia, and improved sprint performance after ischemia-reperfusion likely by increasing glycolytic energy production, as reflected by higher blood lactate concentrations after the sprints. Similar responses were elicited by the two doses tested. In conclusion, acute and prolonged supplementation with mangiferin combined with luteolin enhances performance, muscle O2 extraction, and brain oxygenation during sprint exercise, at high and low doses.

Mangifera indica L. Leaf Extract in Combination With Luteolin or Quercetin Enhances VO2peak and Peak Power Output, and Preserves Skeletal Muscle Function During Ischemia-Reperfusion in Humans.

It remains unknown whether polyphenols such as luteolin (Lut), mangiferin and quercetin (Q) have ergogenic effects during repeated all-out prolonged sprints. Here we tested the effect of Mangifera indica L. leaf extract (MLE) rich in mangiferin (Zynamite®) administered with either quercetin (Q) and tiger nut extract (TNE), or with luteolin (Lut) on sprint performance and recovery from ischemia-reperfusion. Thirty young volunteers were randomly assigned to three treatments 48 h before exercise. Treatment A: placebo (500 mg of maltodextrin/day); B: 140 mg of MLE (60% mangiferin) and 50 mg of Lut/day; and C: 140 mg of MLE, 600 mg of Q and 350 mg of TNE/day. After warm-up, subjects performed two 30 s Wingate tests and a 60 s all-out sprint interspaced by 4 min recovery periods. At the end of the 60 s sprint the circulation of both legs was instantaneously occluded for 20 s. Then, the circulation was re-opened and a 15 s sprint performed, followed by 10 s recovery with open circulation, and another 15 s final sprint. MLE supplements enhanced peak (Wpeak) and mean (Wmean) power output by 5.0-7.0% (P < 0.01). After ischemia, MLE+Q+TNE increased Wpeak by 19.4 and 10.2% compared with the placebo (P < 0.001) and MLE+Lut (P < 0.05), respectively. MLE+Q+TNE increased Wmean post-ischemia by 11.2 and 6.7% compared with the placebo (P < 0.001) and MLE+Lut (P = 0.012). Mean VO2 during the sprints was unchanged, suggesting increased efficiency or recruitment of the anaerobic capacity after MLE ingestion. In women, peak VO2 during the repeated sprints was 5.8% greater after the administration of MLE, coinciding with better brain oxygenation. MLE attenuated the metaboreflex hyperpneic response post-ischemia, may have improved O2 extraction by the Vastus Lateralis (MLE+Q+TNE vs. placebo, P = 0.056), and reduced pain during ischemia (P = 0.068). Blood lactate, acid-base balance, and plasma electrolytes responses were not altered by the supplements. In conclusion, a MLE extract rich in mangiferin combined with either quercetin and tiger nut extract or luteolin exerts a remarkable ergogenic effect, increasing muscle power in fatigued subjects and enhancing peak VO2 and brain oxygenation in women during prolonged sprinting. Importantly, the combination of MLE+Q+TNE improves skeletal muscle contractile function during ischemia/reperfusion.

Uromastyx acanthinura as a natural treatment in a mouse model of type 2 diabetes / Uromastyx acanthinura como tratamiento natural en un modelo murino de diabetes tipo 2

Aims: Oral testimonies from North Africa attribute anti-diabetic effects to medicinal preparations of the lizard Uromastyx acanthinura (UA). No scientific evidence of such effects is currently available. The acute effects of oral administration of UA to C57Bl/6J mice with diet-induced diabetes were tested and, if effectiveness was shown, the effect of subchronic UA administration was assessed in the same model. Methods: Mice were fed a diet containing 60% fat for at least 12 weeks. To assess acute effects, different doses of UA or saline were orally administered with 2 g of glucose/kg during an oral glucose tolerance test (OGTT) on different days in a randomised crossover design. The most effective dose was then fed together with the high-fat diet for 90 days and compared to high-fat diet alone in a parallel design. Body weight (BW), food consumption, welfare, and external appearance were assessed weekly. HbA1c, OGTT, and intraperitoneal insulin tolerance tests (IPITT) were performed at baseline and after treatment. Severity of neuropathy was evaluated by cold allodynia response in the acetone test. Results: UA significantly decreased glucose levels as compared to saline 15 min after administration. After 90 days of treatment, no differences were seen in OGTT or HbA1c between the groups, while IPITT showed higher glucose levels in UA-treated animals. Although weight increase was similar in both groups, weight tended to be higher in the treated group, which had a significantly higher daily food consumption. Cold allodynia response improved in frequency and intensity in the UA group. Conclusions: Orally administered UA acutely decreased blood glucose in diabetic mice. Paradoxically, long-term administration of UA increased food consumption, weight, and insulin resistance. Improved nociceptive response suggested an effect on pain and/or neuropathy. Although additional studies are needed to elucidate the properties and potential applications of UA, our results highlight the value of ethnomedical approaches to African traditional medicine as starting point to evaluate new bioactive components (AU)

Objetivos: Testimonios orales Norteafricanos atribuyen efectos hipoglucemiantes a preparados medicinales del lagarto Uromastyx acanthinura (UA), para los que no existen evidencias científicas actualmente. El objetivo de este trabajo fue el de investigar los efectos agudos de UA administrado oralmente en ratones diabéticos C57Bl/6J inducidos por dieta grasa, y si se demostrase su efectividad evaluar el efecto de su administración subcrónica en el mismo modelo animal. Métodos: Fue administrada una dieta a los animales con un contenido graso del 60% durante al menos 12 semanas. Para evaluar los efectos agudos diferentes dosis de UA o suero salino fueron administrados conjuntamente con 2 g/kg de glucosa durante sobrecargas orales de glucosa (SOG), en diferentes días, siguiendo un diseño cruzado aleatorizado. La dosis más efectiva en esta fase fue entonces administrada mezclada en la dieta durante 90 días y comparada con dieta solo en un diseño paralelo. El peso corporal y el consumo de alimento fueron evaluados semanalmente. HbA1c, SOG, y test de tolerancia intraperitoneal a la insulina (TTIPI) fueron realizados al inicio y tras el tratamiento. La gravedad de la neuropatía fue determinada mediante la evaluación de la alodinia al frío. Resultados: El UA redujo significativamente las concentraciones de glucosa de manera aguda en comparación con el control a los 15 min tras su administración. Tras 90 días de tratamiento no se observaron diferencias en las SOG o HbA1c entre grupos, mientras que para los test de tolerancia intraperitoneal a la isulina valores más altos de glucosa fueron determinados en los animales tratados con UA. Aunque ambos grupos aumentaron su peso, este tendió a ser mayor en los tratados, que a su vez consumieron significativamente más comida por día. La respuesta a la alodinia al frío mejoró en frecuencia e intensidad en los tratados con UA. Conclusiones: El UA administrado oralmente redujo de manera aguda la glucosa en sangre en ratones con diabetes. Paradójicamente, su administración crónica aumentó el consumo de alimento, el peso y la resistencia a la insulina. La mejora en la respuesta nociceptiva sugiere un efecto en el dolor y/o la neuropatía. Aunque son necesarios más estudios para aclarar las propiedades y posibles aplicaciones de este producto, nuestros resultados subrayan el valor de los enfoques etnomédicos hacia la medicina tradicional africana como origen para la evaluación de nuevos compuestos bioactivos (AU)

Uromastyx acanthinura as a natural treatment in a mouse model of type 2 diabetes.

AIMS: Oral testimonies from North Africa attribute anti-diabetic effects to medicinal preparations of the lizard Uromastyx acanthinura (UA). No scientific evidence of such effects is currently available. The acute effects of oral administration of UA to C57Bl/6J mice with diet-induced diabetes were tested and, if effectiveness was shown, the effect of subchronic UA administration was assessed in the same model. METHODS: Mice were fed a diet containing 60% fat for at least 12 weeks. To assess acute effects, different doses of UA or saline were orally administered with 2g of glucose/kg during an oral glucose tolerance test (OGTT) on different days in a randomised crossover design. The most effective dose was then fed together with the high-fat diet for 90 days and compared to high-fat diet alone in a parallel design. Body weight (BW), food consumption, welfare, and external appearance were assessed weekly. HbA1c, OGTT, and intraperitoneal insulin tolerance tests (IPITT) were performed at baseline and after treatment. Severity of neuropathy was evaluated by cold allodynia response in the acetone test. RESULTS: UA significantly decreased glucose levels as compared to saline 15min after administration. After 90 days of treatment, no differences were seen in OGTT or HbA1c between the groups, while IPITT showed higher glucose levels in UA-treated animals. Although weight increase was similar in both groups, weight tended to be higher in the treated group, which had a significantly higher daily food consumption. Cold allodynia response improved in frequency and intensity in the UA group. CONCLUSIONS: Orally administered UA acutely decreased blood glucose in diabetic mice. Paradoxically, long-term administration of UA increased food consumption, weight, and insulin resistance. Improved nociceptive response suggested an effect on pain and/or neuropathy. Although additional studies are needed to elucidate the properties and potential applications of UA, our results highlight the value of ethnomedical approaches to African traditional medicine as starting point to evaluate new bioactive components.

Fertility is reduced in women and in men with type 1 diabetes: results from the Type 1 Diabetes Genetics Consortium (T1DGC).

AIMS/HYPOTHESIS: A recent Finnish study described reduced fertility in patients with childhood-onset type 1 diabetes. The Type 1 Diabetes Genetics Consortium (T1DGC) is an international programme studying the genetics and pathogenesis of type 1 diabetes that includes families with the disease. Our aim was to assess fertility, defined as number of offspring, in the affected and unaffected siblings included in the T1DGC. METHODS: Clinical information from participants aged ≥18 years at the time of examination was included in the present analysis. The number of offspring of affected and unaffected siblings was compared (in families including both) and the influence of birth year, disease duration and age of onset was assessed, the last in affected siblings only, using Poisson regression models. RESULTS: A total of 3010 affected and 801 unaffected adult siblings that belonged to 1761 families were assessed. The mean number of offspring was higher in the unaffected than in the affected individuals, and the difference between the two groups was more pronounced in women than men. Poisson regression analysis showed that both sex and birth cohort significantly affected the differences between groups. In the affected siblings, adult onset (≥18 years), female sex and older birth cohort were associated with higher fertility. CONCLUSIONS/INTERPRETATION: Patients with type 1 diabetes have fewer children than their unaffected siblings. This effect is more evident in women and in older birth cohorts. Onset of type 1 diabetes as an adult rather than a child is associated with a higher number of offspring, even after accounting for birth cohort and disease duration.

Predictors of associated autoimmune diseases in families with type 1 diabetes: results from the Type 1 Diabetes Genetics Consortium.

BACKGROUND: Type 1 diabetes (T1D) is a clinically heterogeneous disease. The presence of associated autoimmune diseases (AAIDs) may represent a distinct form of autoimmune diabetes, with involvement of specific mechanisms. The aim of this study was to find predictors of AAIDs in the Type 1 Diabetes Genetics Consortium data set. METHODS: Three thousand two hundred and sixty-three families with at least two siblings with T1D were included. Clinical information was obtained using questionnaires, anti-GAD (glutamic acid decarboxylase) and anti-protein tyrosine phosphatase (IA-2) were measured and human leukocyte antigen (HLA) genotyping was performed. Siblings with T1D with and without AAIDs were compared and a multivariate regression analysis was performed to find predictors of AAIDs. T1D-associated HLA haplotypes were defined as the four most susceptible and protective, respectively. RESULTS: One or more AAIDs were present in 14.4% of the T1D affected siblings. Age of diabetes onset, current age and time since diagnosis were higher, there was a female predominance and more family history of AAIDs in the group with AAIDs, as well as more frequent anti-GAD and less frequent anti-IA-2 antibodies. Risk and protective HLA haplotype distributions were similar, though DRB1*0301-DQA1*0501-DQB1*0201 was more frequent in the group with AAIDs. In the multivariate analysis, female gender, age of onset, family history of AAID, time since diagnosis and anti-GAD positivity were significantly associated with AAIDs. CONCLUSIONS: In patients with T1D, the presence of AAIDs is associated with female predominance, more frequent family history of AAIDs, later onset of T1D and more anti-GAD antibodies, despite longer duration of the disease. The predominance of certain HLA haplotypes suggests that specific mechanisms of disease may be involved.

A system for repertoire cloning and phage display of murine and leporid antibody fragments.

Even though rabbit antibodies (Abs) are known to exceed murine Abs with respect to specificity, affinity, and stability, cloned leporid immune repertoires have been rarely considered in recombinant Ab preparation for environmental analysis. We have developed a set of four tet(p/o)-based phasmid vectors that allow the efficient cloning of both murine and leporid Ab repertoires. These vectors differ in the design of the cloning sites, choice of signal peptides, and antibiotic selection markers. A set of 39 primer oligodeoxynucleotides has been developed for the PCR amplification of rabbit Ab genes, representing the most exhaustive coverage of the leporid immune repertoire described so far. The atrazine-specific murine Fab fragment K411B and a cloned V-gene repertoire from sulfonamide-immunized rabbits were used to compare these phasmids with respect to expression of Fab fragments, phagemid titers, and number of Fab displaying phagemid particles. Our results show that the ratio of recombinant phagemids could be increased up to 65% of total phage titer by utilizing the appropriate phasmid. Based on this system, the selection of two sulfonamide-specific rabbit Abs, SA2 23 and SA2 90, was accomplished after a single phagemid panning round.

An expression system for the E. coli fermentation of recombinant antibody Fab fragments from mice and rabbits.

A mutually compatible vector system was developed for the bench-top fermenter production of mouse and rabbit Fab fragments comprising pASK85-pro-K411B and pASK85Rab-pro-WR13. These vectors provide a mouse- and rabbit-specific cloning site, respectively, the tetA promoter/operator and the proBA operon that complements the Pro biosynthetic deficiency of the Escherichia coli strain JM83 and can serve as an additional selection marker. Fermentation at elevated cell density (OD600 = 20-40) of the atrazine-specific mouse Fab fragment K411B using JM83 harboring pASK85-pro-K411B in a 2 L bench-top vessel resulted in a yield of 240 microg/L x OD600 affinity-purified protein (13.8 mg). In contrast, expression of leporid Fab fragments using pASK85Rab-pro-WR13 was unsuccessful due to the aggregation of rabbit light chains, which probably relates to a general problem of this specific class of immunoglobulins with their additional Cys residues. Coexpression of rabbit Fab fragments together with four periplasmic folding-helper proteins encoded on pTUM4 led to a significantly improved folding efficiency, resulting in a yield of 50 microg/L x OD600 affinity-purified rabbit Fab fragment (3.3 mg) from the 2 L bench-top fermenter.