Neurotized fasciocutaneous flaps and split-skin grafted muscle flaps are the most frequently used free flap alternatives for the reconstruction of weight-bearing surfaces of the foot. An objective comparison of the innate characteristics of these two flap types, with respect to long-term stability, has not been possible because sensory reinnervation in the fasciocutaneous flaps has been a confounding factor. This study compares nonsensate fasciocutaneous flaps (n = 9) with nonsensate split-skin grafted muscle flaps (n = 11), with mean follow-up periods of 34.3 and 31.3 months, respectively. Patients completed a form that included questions regarding degree of pain at the operative site, presence of ulcers, ability to wear normal shoes, employment status, and time spent standing on foot. Touch and deep sensation were evaluated with Semmes-Weinstein and vibration tests, respectively. Significantly less pain and less ulceration (p < 0.05) were observed in the fasciocutaneous group. Semmes-Weinstein monofilament tests revealed poorer results with split-skin grafted muscle flaps, compared with fasciocutaneous flaps. These results indicate that even if the sensory protection of fasciocutaneous flaps is not considered, these flaps have superior properties, compared with split-skin grafted muscle flaps.

Arginine deiminase (ADI; EC 3.5.3.6) has been studied as a potential antitumor drug for the treatment of arginine-auxotrophic tumors, such as hepatocellular carcinomas (HCCs) and melanomas. Studies with human lymphatic leukemia cell lines confirmed that ADI is an antiangiogenic agent for treating leukemia. The main limitation of ADI from Pseudomonas plecoglossicida (PpADI) lies in its pH-dependent activity profile, its pH optimum is at 6.5. A pH shift from 6.5 to 7.5 results in an approximately 80 % drop in activity. (The pH of human plasma is 7.35 to 7.45.) In order to shift the PpADI pH optimum, a directed-evolution protocol based on an adapted citrulline-screening protocol in microtiter-plate format was developed and validated. A proof of concept for ADI engineering resulted in a pH optimum of pH 7.0 and increased resistance under physiological and slightly alkaline conditions. At pH 7.4, variant M2 (K5T/D44E/H404R) is four times faster than the wild-type PpADI and retains approximately 50 % of its activity relative to its pH optimum, compared to approximately 10 % in the case of the wild-type PpADI.

Saturation mutagenesis was used to generate eleven substitutions of toluene-o-xylene monooxygenase (ToMO) at alpha subunit (TouA) positions F176 and F196 among which nine were novel: F176H, F176N, F176S, F176T, F196A, F196L, F196T, F196Y, F196H, F196I, and F196V. By testing the substrates phenol, toluene, and naphthalene, these positions were found to influence ToMO oxidation activity and regiospecificity. Specifically, TouA variant F176H was identified that had 4.7-, 4.3-, and 1.8-fold faster hydroxylation activity towards phenol, toluene, and naphthalene, respectively, compared to native ToMO. The F176H variant also produced the novel product hydroquinone (61%) from phenol, made twofold more 2-naphthol from naphthalene (34% vs. 16% by the wild-type ToMO), and had the regiospecificity of toluene changed from 51% to 73% p-cresol. The TouA F176N variant had the most para-hydroxylation capability, forming p-cresol (92%) from toluene and hydroquinone (82%) from phenol as the major product, whereas native ToMO formed 30% o-cresol, 19% m-cresol, and 51% of p-cresol from toluene and 100% catechol from phenol. For naphthalene oxidation, TouA variant F176S exhibited the largest shift in the product distribution by producing threefold more 2-naphthol. Among the other F196 variants, F196L produced catechol from phenol two times faster than the wild-type enzyme. The TouA F196I variant produced twofold less o-cresol and 19% more p-cresol from toluene, and the TouA F196A variant produced 62% more 2-naphthol from naphthalene compared to wild-type ToMO. Both of these positions have never been studied through the saturation mutagenesis and some of the best substitutions uncovered here have never been predicted and characterized for aromatics hydroxylation.

Structural analysis of toluene-o-xylene monooxygenase (ToMO) hydroxylase revealed the presence of three hydrophobic cavities, a channel, and a pore leading from the protein surface to the active site. Here, saturation mutagenesis was used to investigate the catalytic roles of alpha-subunit (TouA) second cavity residue L95 and TouA channel residues Q204, D211, and F269. By testing the substrates toluene, phenol, nitrobenzene, and/or naphthalene, these positions were found to influence the catalytic activity of ToMO. Several regiospecific variants were identified from TouA positions Q204, F269, and L95. For example, TouA variant Q204H had the regiospecificity of nitrobenzene changed significantly from 30 to 61 % p-nitrophenol. Interestingly, a combination of mutations at Q204H and A106V altered the regiospecificity of nitrobenzene back to 27 % p-nitrophenol. TouA variants F269Y, F269P, Q204E, and L95D improved the meta-hydroxylating capability of nitrobenzene by producing 87, 85, 82, and 77 % m-nitrophenol, respectively. For naphthalene oxidation, TouA variants F269V, Q204A, Q204S/S222N, and F269T had the regiospecificity changed from 16 to 9, 10, 23, and 25 % 2-naphthol, respectively. Here, two additional TouA residues, S222 and A106, were also identified that may have important roles in catalysis. Most of the isolated variants from D211 remained active, whereas having a hydrophobic residue at this position appeared to diminish the catalytic activity toward naphthalene. The mutational effects on the ToMO regiospecificity described here suggest that it is possible to further fine tune and engineer the reactivity of multicomponent diiron monooxygenases toward different substrates at positions that are relatively distant from the active site.