Identification of two subtilisin-like serine proteases engaged in the degradation of recombinant proteins in Nicotiana benthamiana.

The tobacco variant Nicotiana benthamiana has recently emerged as a versatile host for the manufacturing of protein therapeutics, but the fidelity of many recombinant proteins generated in this system is compromised by inadvertent proteolysis. Previous studies have revealed that the anti-HIV-1 antibodies 2F5 and PG9 as well as the protease inhibitor α1 -antitrypsin (A1AT) are particularly susceptible to N. benthamiana proteases. Here, we identify two subtilisin-like serine proteases (NbSBT1 and NbSBT2) whose combined action is sufficient to account for all major cleavage events observed upon expression of 2F5, PG9 and A1AT in N. benthamiana. We propose that downregulation of NbSBT1 and NbSBT2 activities could constitute a powerful means to optimize the performance of this promising platform for the production of biopharmaceuticals. DATABASES: NbSBT sequence data are available in the DDBJ/EMBL/GenBank databases under the accession numbers MN534996 to MN535005.

Steric Accessibility of the Cleavage Sites Dictates the Proteolytic Vulnerability of the Anti-HIV-1 Antibodies 2F5, 2G12, and PG9 in Plants.

Broadly neutralizing antibodies (bNAbs) to human immunodeficiency virus type 1 (HIV-1) hold great promise for immunoprophylaxis and the suppression of viremia in HIV-positive individuals. Several studies have demonstrated that plants as Nicotiana benthamiana are suitable hosts for the generation of protective anti-HIV-1 antibodies. However, the production of the anti-HIV-1 bNAbs 2F5 and PG9 in N. benthamiana is associated with their processing by apoplastic proteases in the complementarity-determining-region (CDR) H3 loops of the heavy chains. Here, it is shown that apoplastic proteases can also cleave the CDR H3 loop of the bNAb 2G12 when the unusual domain exchange between its heavy chains is prevented by the replacement of Ile19 with Arg. It is demonstrated that CDR H3 proteolysis leads to a strong reduction of the antigen-binding potencies of 2F5, PG9, and 2G12-I19R. Inhibitor profiling experiments indicate that different subtilisin-like serine proteases account for bNAb fragmentation in the apoplast. Differential scanning calorimetry experiments corroborate that the antigen-binding domains of wild-type 2G12 and 4E10 are more compact than those of proteolysis-sensitive antibodies, thus shielding their CDR H3 regions from proteolytic attack. This suggests that the extent of proteolytic inactivation of bNAbs in plants is primarily dictated by the steric accessibility of their CDR H3 loops.

Endoscopic submucosal dissection training with pig models in a Western country.

AIM: To test a strategy for endoscopic submucosal dissection (ESD) training in animal models designed to overcome the initial learning curve. METHODS: ESD was attempted in ex vivo and in vivo pig models. Thirty ESD procedures were attempted in the esophagus (n = 9) or the stomach (n = 21). The ex vivo model was used until initial competence was achieved. In the in vivo model, several ESD procedures were performed in up to 3 sessions. The following variables were analyzed: specimen size, complete and en bloc resection rate, time for circumferential incision, time for submucosal dissection, total ESD duration, and complications. RESULTS: Complete resection was achieved in 28 cases (en bloc 27); 2 could not be completed (one perforation, one technical difficulty). The mean +/- SD time for circumferential incision was 36.2 +/- 16.8 min (range: 8-87 min), and the mean +/- SD time for submucosal dissection was 45.1 +/- 35.7 min (range: 9-196 min). The mean +/- SD size of the resected specimens was 45.2 +/- 17.8 mm. The mean +/- SD total resection time was significantly increased for the gastric cases performed in the first half of the study (n = 13) than in the second half (n = 8) (98.9 +/- 62.4 min vs 61.7 +/- 17.6 min, P = 0.04), although the specimen size did not differ. CONCLUSION: Training in animal models could help endoscopists overcome the learning curve before starting ESD in humans.

Sural and radial sensory responses in healthy adults: diagnostic implications for polyneuropathy.

We prospectively performed sural and radial sensory nerve conduction studies in 92 healthy subjects, aged between 21 and 88 years, both to determine the lower limits of normal (LLN) and to assess the effects of age and body mass index (BMI) on the sural and radial sensory nerve action potential (SNAP) amplitudes and on the sural/radial amplitude ratio (SRAR). Using the nonparametric bootstrap method to calculate 95% confidence intervals, we found that the 5% LLN values for sural and radial SNAPs were 14 microV and 25.5 microV in subjects aged < or =39 years, 7 microV and 17.4 microV in subjects aged 40-59 years, and 3 microV and 12 microV in subjects aged > or =60 years. The 5% LLN for SRAR for all patients was 0.21. Sural and radial SNAP amplitudes but not SRAR were strongly and inversely correlated with age and BMI. These age-adjusted normal values and revised SRAR will aid in the electrodiagnosis of polyneuropathy.

Ulnar neuropathy in the forearm: A possible complication of diabetes mellitus.

Ulnar neuropathy in the forearm is an unusual cause of hand weakness and sensory loss that is most often attributed to compression of the nerve distally within the humero-ulnar arcade (cubital tunnel). An association with diabetes mellitus, however, has not been reported. We identified four patients with type I diabetes mellitus and clinical findings suggestive of ulnar neuropathy in whom electrophysiologic testing revealed partial conduction block or abnormal temporal dispersion within the forearm segment of the ulnar nerve. Although evidence for a mild underlying polyneuropathy was present in three patients, the ulnar nerve abnormalities were disproportionately severe. In all cases, a Martin-Grüber anastomosis was excluded. Whether this lesion is due to an increased propensity to focal compression of the ulnar nerve within the humero-ulnar arcade or whether it represents a localized manifestation of the generalized polyneuropathy remains to be determined.

Perfil patológico de obreros portuarios pensionados dominicanos / Pathologic profile of Dominican port workers pensioner

Estudio descriptivo retrospectivo, con una fase de observacion participante de las condiciones de trabajo y una fase de recoleccion retrospectiva de la informacion, realizado con el objetivo de determinar el perfil patologico de 269 obreros portuarios pensionados por trastornos de salud, Santo Domingo, Republica Dominicana. La morbimortalidad fue distribuida por sistemas, siendo el mas afectado el sistema osteomuscular con un 30.7 por ciento (175 casos), siendo el desgaste fisico la patologia mas frecuente dentro de este sistema. El segundo sistema mas afectado es el cardiovacuslar con un 16.7 por ciento (95 casos) siendo la hipertension arterial de un 70.5 por ciento (67 casos). Los obreros portuarios mas afectados fueron los carretilleros, bodegueros y estibadores, cuyos puestos de trabajo les someten a mayor deterioro en su salud que en los puestos de trabajo limitados solo a la supervision como son los capataces, ayudantes y chequeadores