Nuclear heterogeneity is prevalent in high-quality fractionated human sperm cells typically used for assisted conception.

STUDY QUESTION: What is the nuclear heterogeneity of high-density purified human spermatozoa typically used for IVF purposes. SUMMARY ANSWER: The data show that while density gradient separation has improved the overall sperm population, there is still a large degree of nuclear heterogeneity within these cells. WHAT IS KNOWN ALREADY: Chromomycin A3 (CMA3) is an important DNA binding fluorochrome for the assessment of male-factor fertility. It is typically used to predict IVF outcomes on entire sperm ejaculates with very high receiver operating characteristic. Here we used CMA3 to characterise typical populations of human spermatozoa that would be used for IVF purposes after density gradient separation. STUDY DESIGN, SIZE, DURATION: We compared the intensity of CMA3 binding within high-dense sperm populations obtained from men. Binding heterogeneity was confirmed through fluorescence microscopy and FACS analysis independently. We also looked at CMA3 staining directly with head morphology in this sperm population. Finally, we looked at electron micrographs of nuclear heterogeneity (vacuoles, chromatin compaction) of spermatozoa following density gradient sorting of CMA3-stained cells. PARTICIPANTS/MATERIALS, SETTING, METHODS: We used sperm donors who had fathered one or more children. Semen was collected after 2 days abstinence and purified over Percoll gradients. Only the high-quality spermatozoa, the same used for assisted conception, were then used. Cells were stained with CMA3 and sorted using FACS. Following this, electron micrographs were used to assess nuclear heterogeneity of CMA3-dependent sorted spermatozoa. MAIN RESULTS AND THE ROLE OF CHANCE: CMA3 staining occurs within morphologically normal as well as abnormal spermatozoa. High-intensity CMA3-stained sperm possessed large vacuoles that were not seen in the low-CMA3 population. In addition, the high-CMA3 stained cells possess higher amounts of nuclear granulation. LIMITATIONS, REASONS FOR CAUTION: The present study only describes the issues within the chromatin of these cells and does not suggest an alternate selection technique. WIDER IMPLICATIONS OF THE FINDINGS: CMA3 is one of the better reported prognostic assays in predicting pregnancy outcomes, especially in cases where the male is at fault. However, it is clear that even in fractionated populations of human spermatozoa, there are sperm cells that are morphologically normal yet possess high levels of CMA3 staining and chromatin granulation. The implication of this is that the embryologist, whom selects on the basis of sperm morphology, may choose a cell with poor chromatin, which may lead to poor embryo outcomes. STUDY FUNDING/COMPETING INTEREST(S): The project was funded by the National Health and Medical Research council, APP1118943. The authors have no conflict of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

HIPECT4: multicentre, randomized clinical trial to evaluate safety and efficacy of Hyperthermic intra-peritoneal chemotherapy (HIPEC) with Mitomycin C used during surgery for treatment of locally advanced colorectal carcinoma.

BACKGROUND: Local relapse and peritoneal carcinomatosis (PC) for pT4 colon cancer is estimated in 15,6% and 36,7% for 12 months and 36 months from surgical resection respectively, achieving a 5 years overall survival of 6%. There are promising results using prophylactic HIPEC in this group of patients, and it is estimated that up to 26% of all T4 colon cancer could benefit from this treatment with a minimal morbidity. Adjuvant HIPEC is effective to avoid the possibility of peritoneal seeding after surgical resection. Taking into account these results and the cumulative experience in HIPEC use, we will lead a randomized controlled trial to determine the effectiveness and safety of adjuvant treatment with HIPEC vs. standard treatment in patients with colon cancer at high risk of peritoneal recurrence (pT4). METHODS/DESIGN: The aim of this study is to determine the effectiveness and safety of adjuvant HIPEC in preventing the development of PC in patients with colon cancer with a high risk of peritoneal recurrence (cT4). This study will be carried out in 15 Spanish HIPEC centres. Eligible for inclusion are patients who underwent curative resection for cT4NxM0 stage colon cancer. After resection of the primary tumour, 200 patients will be randomized to adjuvant HIPEC followed by routine adjuvant systemic chemotherapy in the experimental arm, or to systemic chemotherapy only in the control arm. Adjuvant HIPEC will be performed simultaneously after the primary resection. Mitomycin C will be used as chemotherapeutic agent, for 60 min at 42-43 °C. Primary endpoint is loco-regional control (LC) in months and the rate of loco-regional control (%LC) at 12 months and 36 months after resection. DISCUSSION: We assumed that adjuvant HIPEC will reduce the expected absolute risk of peritoneal recurrence from 36% to 18% at 36 months for T4 colon-rectal carcinoma. TRIAL REGISTRATION: NCT02614534 ( clinicaltrial.gov ) Nov-2015.

Characteristics and treatment of pyoderma gangrenosum in inflammatory bowel disease.

BACKGROUND: Pyoderma gangrenosum is a serious cutaneous complication seen in approximately 1 % of patients with inflammatory bowel disease (IBD). Oral corticosteroids are the mainstay treatment, although the evidence supporting their use is weak. AIMS: The purpose of this study was to investigate the characteristics of pyoderma gangrenosum associated with Crohn's disease or ulcerative colitis and which treatments are prescribed in Spanish clinical practice. METHODS: In this retrospective, observational study, the medical records from all patients with IBD and a diagnosis of pyoderma gangrenosum attended by the gastroenterology departments of 12 Spanish hospitals were reviewed. Data on patient demographics and characteristics, underlying IBD and treatment, and pyoderma gangrenosum characteristics, treatment, and outcome were collected and analyzed. RESULTS: The data from 67 patients were analyzed (41 [61.2 %] women, 41 [61.2 %] with Crohn's disease, 25 [37.3 %] with ulcerative colitis, and 1 [1.5 %] with indeterminate disease). The underlying disease was in remission in approximately one-third of patients at the time of presentation of pyoderma gangrenosum. Healing was achieved in all patients (in 3 without any systemic therapy). Oral corticosteroids were taken by 51 patients (76.1 %), almost always as first-line treatment, although definitive healing was attained in 19 (28.4 %). Biologic agents such as infliximab and adalimumab were taken by 31 patients (46.3 %) at some point (first-line in 6 patients [9.0 %]), with definitive healing in 29 patients (93.5 %). CONCLUSIONS: Oral corticosteroid therapy remains the most common treatment for pyoderma gangrenosum associated with inflammatory bowel disease. Biologic therapies such as infliximab and adalimumab should also be considered.

Minimally invasive treatment of colorectal liver metastases.

Minimally invasive approaches are increasingly used in the treatment of colorectal liver metastases (CRLMs) and for increasingly complex cases. Herein, we review the spectrum of modalities used in the minimally invasive treatment of CRLM, focusing on one of the newest approaches, robotic liver resection, and on local regional therapies, both operative and percutaneous. Oncologic outcomes after minimally invasive therapies are also evaluated. Although there are no randomized trials comparing minimally invasive liver resection to open resection, an increasing quantity of nonrandomized data suggest favorable outcomes with a minimally invasive approach. The future of minimally invasive treatment of CRLM will likely include specifying training and credentialing criteria as well as an enlarging role for the combined surgical treatment of CRLM and extrahepatic colorectal metastases.

Global optimization in systems biology: stochastic methods and their applications.

Mathematical optimization is at the core of many problems in systems biology: (1) as the underlying hypothesis for model development, (2) in model identification, or (3) in the computation of optimal stimulation procedures to synthetically achieve a desired biological behavior. These problems are usually formulated as nonlinear programing problems (NLPs) with dynamic and algebraic constraints. However the nonlinear and highly constrained nature of systems biology models, together with the usually large number of decision variables, can make their solution a daunting task, therefore calling for efficient and robust optimization techniques. Here, we present novel global optimization methods and software tools such as cooperative enhanced scatter search (eSS), AMIGO, or DOTcvpSB, and illustrate their possibilities in the context of modeling including model identification and stimulation design in systems biology.

Dual Effect of low-level laser therapy (LLLT) on the acute lung inflammation induced by intestinal ischemia and reperfusion: Action on anti- and pro-inflammatory cytokines.

BACKGROUND AND OBJECTIVE: It is unknown if pro- and anti-inflammatory mediators in acute lung inflammation induced by intestinal ischemia and reperfusion (i-I/R) can be modulated by low-level laser therapy (LLLT). STUDY DESIGN/MATERIAL AND METHODS: A controlled ex vivo study was developed in which rats were irradiated (660 nm, 30 mW, 0.08 cm² of spot size) on the skin over the right upper bronchus 1 hour post-mesenteric artery occlusion and euthanized 4 hours later. For pretreatment with anti-tumor necrosis factor (TNF) or IL-10 antibodies, the rats received either one of the agents 15 minutes before the beginning of reperfusion. METHODS: Lung edema was measured by the Evans blue extravasation and pulmonary neutrophils influx was determined by myeloperoxidase (MPO) activity. Both TNF and IL-10 expression and protein in lung were evaluated by RT-PCR and ELISA, respectively. RESULTS: LLLT reduced the edema (80.1 ± 41.8 µg g⁻¹ dry weight), neutrophils influx (0.83 ± 0.02 × 106 cells ml⁻¹), MPO activity (2.91 ± 0.60), and TNF (153.0 ± 21.0 pg mg⁻¹ tissue) in lung when compared with respective control groups. Surprisingly, the LLLT increased the IL-10 (0.65 ± 0.13) in lung from animals subjected to i-I/R. Moreover, LLLT (0.32 ± 0.07 pg ml⁻¹) reduced the TNF-α level in RPAECs when compared with i-I/R group. The presence of anti-TNF or IL-10 antibodies did not alter the LLLT effect on IL-10 (465.1 ± 21.0 pg mg⁻¹ tissue) or TNF (223.5 ± 21.0 pg mg⁻¹ tissue) in lung from animals submitted to i-I/R. CONCLUSION: The results indicate that the LLLT attenuates the i-I/R-induced acute lung inflammation which favor the IL-10 production and reduce TNF generation.

Low-level laser therapy (LLLT) acts as cAMP-elevating agent in acute respiratory distress syndrome.

The aim of this work was to investigate if the low-level laser therapy (LLLT) on acute lung inflammation (ALI) induced by lipopolysaccharide (LPS) is linked to tumor necrosis factor (TNF) in alveolar macrophages (AM) from bronchoalveolar lavage fluid (BALF) of mice. LLLT has been reported to actuate positively for relieving the late and early symptoms of airway and lung inflammation. It is not known if the increased TNF mRNA expression and dysfunction of cAMP generation observed in ALI can be influenced by LLLT. For in vivo studies, Balb/c mice (n = 5 for group) received LPS inhalation or TNF intra nasal instillation and 3 h after LPS or TNF-α, leukocytes in BALF were analyzed. LLLT administered perpendicularly to a point in the middle of the dissected bronchi with a wavelength of 660 nm and a dose of 4.5 J/cm(2). The mice were irradiated 15 min after ALI induction. In vitro AM from mice were cultured for analyses of TNF mRNA expression and protein and adenosine3':5'-cyclic monophosphate (cAMP) levels. One hour after LPS, the TNF and cAMP levels in AM were measured by ELISA. RT-PCR was used to measure TNF mRNA in AM. The LLLT was inefficient in potentiating the rolipram effect in presence of a TNF synthesis inhibitor. LLLT attenuated the neutrophil influx and TNF in BALF. In AM, the laser increased the cAMP and reduced the TNF-α mRNA. LLLT increases indirectly the cAMP in AM by a TNF-dependent mechanism.

Potential of MMP-9 based nanoparticles at optimizing the cow dry period: pulling apart the effects of MMP-9 and nanoparticles.

The cow dry period is a non-milking interval where the mammary gland involutes and regenerates to guarantee an optimal milk production in the subsequent lactation. Important bottlenecks such as the high risk of intramammary infections complicate the process. Antibiotics have been routinely used as a preventive treatment but the concerns about potential antibiotic resistance open a new scenario in which alternative strategies have to be developed. Matrix metalloproteinase-9 (MMP-9) is an enzyme able to degrade the extracellular matrix, triggering the involution and immune function of cow mammary gland. We have studied the infusion into the mammary gland of MMP-9 inclusion bodies as protein-based nanoparticles, demonstrating that 1.2 mg of MMP-9 enhanced the involution and immune function of the cow mammary gland. However, the comparison of the effects triggered by the administration of an active and an inactive form of MMP-9 led to conclude that the response observed in the bovine mammary gland was mainly due to the protein format but not to the biological activity of the MMP-9 embedded in the inclusion body. This study provides relevant information on the future use of protein inclusion bodies in cow mammary gland and the role of MMP-9 at dry-off.

Lung inflammation and endothelial cell damage are decreased after treatment with phototherapy (PhT) in a model of acute lung injury induced by Escherichia coli lipopolysaccharide in the rat.

Lipopolysaccharide (LPS) mimics the symptoms of acute lung injury (ALI), which is characterized by the accumulation in the lungs of neutrophils producing inflammatory mediators. Because of the lack of information about phototherapy (PhT) effects on ALI, we investigated whether PhT (685nm InGaAlP) attenuates LPS-induced ALI. PhT reduced lung edema, the accumulation of TNF-alpha in the lung, and myeloperoxidase (MPO) activity. However, PhT was not efficient in reducing of TNF-alpha concentration in both serum and neutrophils of blood after LPS. In another series of experiments, in vitro assays of the effects of PhT effect on mouse pulmonary arterial endothelium cells (MPAECs) after TNF-alpha showed that the laser restores the MPAECs damage induced at 6 or 24h after TNF-alpha. These results suggest the PhT effect on ALI is partly due to inhibition of TNF-alpha release from neutrophils and lung cells.

Effect of progesterone and ionomycin on domestic cat sperm motility patterns and acrosome reaction.

This study was aimed at assessing the changes in sperm motion patterns and the percentage of acrosome reaction (AR) in domestic cat semen after treatment with either ionomycin or progesterone (P(4)). Ten ejaculates were collected from five tomcats using an artificial vagina, and were diluted, centrifuged and resuspended in a capacitation medium. Samples were evaluated and divided into seven equal aliquots and, after 2 h at 25 degrees C, were incubated for 30 min at 38 degrees C in 5% CO(2) and then analyzed. Computer-assisted sperm analysis and a combination of three fluorescent probes were used to assess sperm plasma, acrosomal membrane integrity and mitochondrial transmembrane potential. Thirty minutes after the start of incubation, P(4) was added (10 microg/ml) to the P1 group. Groups P2 and P3 were supplemented with P(4) (10 and 20 microg/ml, respectively) only after 2 h of incubation, and groups I1 and I2 were supplemented with ionomycin (4 and 8 mum, respectively) 2 h after incubation. Group E was supplemented with ethanol (0.6%) at 2 h after incubation and group C received no supplementation. Ionomycin and P(4) treatments led to a hyperactivation-like sperm motion and an increase (p < 0.05) in the percentage of AR. Although a higher (p < 0.05) percentage of AR was obtained in group I2 when compared with all P(4) groups, a decrease (p < 0.05) in total and progressive motility was observed in I2 group. As I1 group was similar to I2 to induce AR without diminishing sperm motility, we can conclude that ionomycin at 4 microm seems to be more suitable to trigger AR in domestic cat sperm.

Impact of 24-h cooling prior to freezing on the survival of domestic cat (Felis catus) epididymal sperm.

The aim of this study was to investigate the impact of a 24-h cooling period prior to freezing on domestic cat epididymal sperm viability. Fifteen tomcats were submitted to routine orchiectomy and sperm samples were retrieved from both epididymides in a Tris-glucose-20% egg yolk extender. For each tomcat, the diluted sperm was split into two equal volumes and cooled to 5 degrees C at a rate of 0.5 degrees C/min; one sample for 60 min (control) and the other for 24 h (cooled). After the cooling period, samples from both groups were frozen using an identical freezing protocol. Sperm samples were evaluated in three different periods: immediately after harvesting, after cooling at 5 degrees C for 24 h (cooled group) and after freezing-thawing of control and cooled groups. Evaluations consisted of sperm motility and progressive status, sperm morphology and plasma membrane integrity (PMI) using two fluorescent probes. After cooling for 24 h, a decrease (p < 0.05) in sperm motility, progressive status and PMI was observed when compared to sperm samples immediately after collection. Comparing the results obtained after thawing, no difference (p < 0.05) was found regarding sperm motility, progressive status, PMI and sperm morphology between control and cooled groups. The results from the present study show that cooling cat epididymal spermatozoa at 5 degrees C for 24 h prior to freezing does not lead to major damage of spermatozoa impairing the freeze-thaw process.

[Rare diseases. Concept, epidemiology and state of the question in Spain]. / Enfermedades raras. Concepto, epidemiología y situación actual en España.

Rare diseases are those whose prevalence is below 5 cases per 10,000 inhabitants in the European Community. Most cases are diagnosed during paediatric age due to their genetic origin, while some others are congenital malformations. Nevertheless, a higher prevalence is seen during adulthood as most of the former diseases are very severe and patients die during childhood. At the same time, higher survival rates are related to some chronic rare diseases in adults. The Spanish Network of Research Epidemiology for Rare Diseases (REpIER) developed the first atlas showing the geographical distribution of rare diseases in Spain, assessed the Spanish rare disease registries, contributed to the further development of regional plans on rare diseases, as well as to social and health actions, and established the real group of needs to be solved. These have been included in the Communication of the European Commission on Rare Diseases as well as in the Spanish Senate Presentation.

A new approach to obtain pure and active proteins from Lactococcus lactis protein aggregates.

The production of pure and soluble proteins is a complex, protein-dependent and time-consuming process, in particular for those prone-to-aggregate and/or difficult-to-purify. Although Escherichia coli is widely used for protein production, recombinant products must be co-purified through costly processes to remove lipopolysaccharide (LPS) and minimize adverse effects in the target organism. Interestingly, Lactococcus lactis, which does not contain LPS, could be a promising alternative for the production of relevant proteins. However, to date, there is no universal strategy to produce and purify any recombinant protein, being still a protein-specific process. In this context and considering that L. lactis is also able to form functional protein aggregates under overproduction conditions, we explored the use of these aggregates as an alternative source of soluble proteins. In this study, we developed a widely applicable and economically affordable protocol to extract functional proteins from these nanoclusters. For that, two model proteins were used: mammary serum amyloid A3 (M-SAA3) and metalloproteinase 9 (MMP-9), a difficult-to-purify and a prone-to-aggregate protein, respectively. The results show that it is possible to obtain highly pure, soluble, LPS-free and active recombinant proteins from L. lactis aggregates through a cost-effective and simple protocol with special relevance for difficult-to-purify or highly aggregated proteins.

Anti-inflammatory effects of low-level laser therapy (LLLT) with two different red wavelengths (660 nm and 684 nm) in carrageenan-induced rat paw edema.

It has been suggested that low-level laser therapy (LLLT) can modulate inflammatory processes. The aim of this experiment was to investigate what effects red laser irradiation with two different wavelengths (660 nm and 684 nm) on carrageenan-induced rat paw edema and histology. Thirty two male Wistar rats were randomly divided into four groups. One group received a sterile saline injection, while inflammation was induced by a sub-plantar injection of carrageenan (1 mg/paw) in the three other groups. After 1 h, LLLT was administered to the paw in two of the carrageenan-injected groups. Continuous wave 660 nm and 684 nm red lasers respectively with mean optical outputs of 30 mW and doses of 7.5 J/cm(2) were used. The 660 nm and 684 nm laser groups developed significantly (p<0.01) less edema (0.58 ml [SE+/-0.17] ml and 0.76 ml [SE+/-0.10] respectively) than the control group (1.67 ml [SE+/-0.19]) at 4h after injections. Similarly, both laser groups showed a significantly lower number of inflammatory cells in the muscular and conjunctive sub-plantar tissues than the control group. We conclude that both 660 nm and 684 nm red wavelengths of LLLT are effective in reducing edema formation and inflammatory cell migration when a dose of 7.5 J/cm(2) is used.

Efectividad y seguridad de pirfenidona en la fibrosis pulmonar idiopática / Effectiveness and safety of pirfenidone in idiopathic pulmonary fibrosis

Objetivo: La fibrosis pulmonar idiopática (FPI) es una enfermedad caracterizada por una cicatrización progresiva de los pulmones provocando su deterioro a largo plazo. Su diagnóstico requiere la existencia del patrón radiológico denominado neumonía intersticial usual (NIU). Su prevalencia es mayor en hombres. Entre los síntomas principales encontramos la disnea y la tos. En la actualidad, no existe una cura conocida, sin embargo, existen diversos tratamientos antifibróticos enfocados a disminuir la progresión de esta patología y prolongar la supervivencia. El objetivo es evaluar la efectividad y seguridad de la pirfenidona en esta enfermedad.Métodos: Estudio descriptivo retrospectivo entre enero de 2016 y diciembre de 2019. Se incluyeron 40 pacientes diagnosticados de fibrosis pulmonar idiopática que iniciaron tratamiento con pirfenidona.Resultados: Los parámetros de función pulmonar mostraron valores mantenidos durante el periodo de estudio. Hubo mejoría de la tos. Las reacciones adversas más comunes fueron alteraciones gastrointestinales y cutáneas.Conclusión: El tratamiento con pirfenidona requiere de un riguroso seguimiento por el porcentaje de pacientes que abandonó esta terapia. (AU)

Objective: Idiopathic pulmonary fibrosis (IPF) is a disease characterized by progressive scarring of the lungs causing their long-term deterioration. Its diagnosis requires the existence of the radiological pattern called usual interstitial pneumonia (UIP). Its prevalence is higher in men. Among the main symptoms, we find dyspnea and cough. At present, there is no known cure, however, there are various antifibrotic treatments focused on slowing the progression of this pathology and prolonging survival. The aim of this study was to evaluate the effectiveness and safety of pirfenidone in this disease.Methods: Retrospective descriptive study from January 2016 to December 2019. The study included 40 patients diagnosed with idiopathic pulmonary fibrosis who started treatment with pirfenidone.Results: The pulmonary function parameters showed values that were maintained during the study period. There was an improvement in the cough. The most common adverse reactions were gastrointestinal and skin disorders.Conclusion: Pirfenidone treatment requires rigorous monitoring due to the percentage of patients who abandoned this therapy. (AU)

The fusogenic peptide HA2 impairs selectivity of CXCR4-targeted protein nanoparticles.

We demonstrate here that the genetic incorporation of the fusogenic peptide HA2 into a CXCR4-targeted protein nanoparticle dramatically reduces the specificity of the interaction between nanoparticles and cell receptors, a factor to be considered when designing tumor-homing drug vehicles displaying endosomal-escape agents. The loss of specificity is concomitant with enhanced cell penetrability.

Morphological and functional characteristics of chilled semen obtained from domestic feline epididymides (Felis catus).

The objective of this study was to determine morphological and functional characteristics of semen retrieved from the feline epididymis before and after cooling. Sixteen adult male cats were orchiectomized. The distal portion of the epididymis and proximal part of the deferent ducts were dissected and squeezed to obtain their content. After centrifugation, the supernatant was removed, sperm were resuspended in a 0.9 mL Tris-fructose-citric acid extender containing 20% egg yolk, aliquoted into three 0.3 mL samples, placed in a refrigerator (4.8 degrees C) and cooled (0.5 degrees C/min). Semen evaluations were performed on four occassions: immediately after epididymal sperm retrieval (T0), and at 24 h (T1), 48 h (T2) and 72 h (T3) after cooling. On each occasion, progressive motility, vigor and sperm morphology were determined. Mean motility and vigor decreased (P < 0.05) between each successive examination. Although the majority of sperm cell damage occurred within the first 24 h, there was a decrease (P < 0.05) in mean percentage of morphologically normal sperm between T0 and each evaluated time (T1, T2, T3) after cooling, due to an increase in coiled and bent sperm tails. Further studies are needed to evaluate the effects of cooling on the fertilizing capacity of cat epididymal spermatozoa in assisted reproduction programs.

Proteolytic digestion of bacterial inclusion body proteins during dynamic transition between soluble and insoluble forms.

Inclusion bodies formed by two closely related hybrid proteins, namely VP1LAC and LACVP1, have been compared during their building in Escherichia coli. Features of these proteins are determinant of aggregation rates and protein composition of the bodies, generating insoluble particles with distinguishable volume evolution. Interestingly, in LACVP1 and less perceptibly in VP1LAC bodies, an important fraction of the aggregated polypeptide is lost at a given stage of body construction. Stable degradation intermediates of the more fragile LACVP1 are concomitantly found embedded in the bodies. When recombinant protein synthesis is arrested in growing cells, the amount of aggregated protein drops while the amount of soluble protein undergoes a sudden rise before proteolysis. This indicates an architectural plasticity during the in vivo building of the studied inclusion bodies by a dynamic transition between soluble and insoluble forms of the recombinant proteins involved. During this transition, protease-sensitive polypeptides can suffer an efficient proteolytic attack and the resulting fragments further aggregate as inclusion body components.

Reversible activation of a cryptic cleavage site within E. coli beta-galactosidase in beta-galactosidase fusion proteins.

The VP60 capsid protein of rabbit haemorrhagic disease virus (60 kDa) has been fused to the C-terminus of beta-galactosidase and produced in E. coli from two related expression vectors. One of these vectors, carries a 429 bp DNA segment encoding the N-terminus peptide of VP60, and directs the synthesis of a larger fusion that contains the entire viral protein. Both fusion proteins are efficiently cleaved at a presumed trypsin-like target site within the carboxy moiety of beta-galactosidase (Arg 611-Thr 612), which is activated by the presence of the viral partner. In the larger fusion, VP60 is released by a cleavage within the linker region that affects about 10% of the chimeric proteins. In this situation, the resulting beta-galactosidase-like fragment recovers its natural proteolytic stability. These results prove that cryptic cleavage sites in beta-galactosidase can be efficiently activated in a fusion protein and suggest that this activation is based on reversible steric constraints generated by the fusion partner.

3D gene of foot-and-mouth disease virus. Conservation by convergence of average sequences.

The nucleotide sequence of the 3D (polymerase) gene of eight epidemiologically related isolates of foot-and-mouth disease virus of serotype C1 is reported. The genetic heterogeneity of 3D RNA is compared with that of the VP1-coding RNA of the same viruses. Regression lines of substitutions per nucleotide that distinguish any pair of viruses as a function of the time interval between the corresponding isolations show: (1) the slope (substitutions/nucleotide per month) is 2.1 times larger for the VP1 RNA than for the 3D RNA region; (2) the intercept with the ordinate (substitutions/nucleotide) for VP1 RNA is indistinguishable from that for 3D RNA. Thus, the average heterogeneity of the VP1-coding region is very similar to that of the 3D-coding region only among co-circulating viruses. Nine mutations and points of heterogeneity occurred within nucleotide residues 883 to 1026, which encode an amino acid segment, extremely conserved among many different RNA viruses. The results suggest that, rather than due to inherently lower mutability, the conservation of 3D genes is caused by a limitation in the fixation of substitutions in viable genomes.