Nitric oxide (NO) has gained interest as a major signaling molecule during plant development and in response to environmental cues. Formation of NO during symbiotic interactions has been reported, but the role and sources of NO in nodules remain unclear. In this work, the involvement of denitrification, performed by the symbiont Bradyrhizobium japonicum, in NO formation in soybean nodules in response to flooding conditions has been investigated by inoculating plants with napA-, nirK-, or norC-deficient mutants. Levels of nitrosylleghemoglobin (LbNO) in flooded nirK and norC nodules were significantly higher than those observed in wild-type nodules. In addition, nirK and norC nodules accumulated more nitrite and NO, respectively, than wild-type nodules. By contrast, levels of LbNO, nitrite, and NO in flooded napA nodules were lower than in wild-type nodules. These results suggest that LbNO formation in soybean nodules in response to flooding conditions is caused by nitrite and NO generated from periplasmic nitrate reductase (Nap) and also containing nitrite reductase (NirK) denitrification enzymes. Flooding caused a decrease of nifH expression and nitrogenase activity in wild-type and norC nodules but not in napA or nirK nodules. Incubation of wild-type and norC nodules with a NO scavenger counteracted the effect of flooding. Under free-living conditions, β-galactosidase activity from a nifD′-′lacZ fusion decreased in a norC mutant, which also accumulated NO in the medium. These results suggest that NO formed by Cu-containing nitrite reductase in soybean nodules in response to flooding has a negative effect on expression of nitrogenase. We propose that Lb has a major role in detoxifying NO and nitrite produced by bacteroidal denitrification in response to flooding conditions.

It is becoming recognized that leghaemoglobin constitutes an important buffer for the cytotoxic nitric oxide radical (NO•) in root nodules, although the sources of this NO• within nodules are unclear. In Bradyrhizobium japonicum bacteroids, NO• can be produced through the denitrification process, during which nitrate is reduced to nitrite by the periplasmic nitrate reductase Nap, and nitrite is reduced to NO• by the respiratory nitrite reductase NirK. To assess the contribution of bacteroidal denitrification to the NO• within nitrate-treated soybean nodules, electron paramagnetic resonance and UV–visible spectroscopy were employed to study the presence of nitrosylleghaemoglobin (LbNO) within nodules from plants inoculated with wild-type, napA or nirK B. japonicum strains. Since it has been found that hypoxia induces NO• production in plant root tissue, and that plant roots can be subjected to hypoxic stress during drought and flooding, the effect of hypoxic stress on the formation of LbNO complexes within nodules was also investigated. Maximal levels of LbNO were observed in nodules from plants treated with nitrate and subjected to hypoxic conditions. It is shown that, in the presence of nitrate, all of the LbNO within normoxic nodules arises from nitrate reduction by the bacteroidal periplasmic nitrate reductase, whereas Nap activity is only responsible for half of the LbNO within hypoxic nodules. In contrast to Nap, NirK is not essential for LbNO formation under any condition tested.

Empirical data on the transfer and persistence of trace DNA are crucial to the evaluation of forensic DNA evidence. This evaluation can be complicated by the occurrence of indirect DNA transfer; the possibility of which is well established, but research into such transfer is often focussed on unrealistic situations, e.g. handling of DNA-free items after participants have shaken hands for 1-2min. To simulate more realistic scenarios, this study investigated the deposition and persistence of both directly- and indirectly-transferred DNA on knives that had been artificially set up as 'regularly-used'. Each knife was handled in a prescribed manner by a specific participant over two consecutive days to simulate regular use. Each participant then shook hands for 10s with a fellow volunteer and immediately stabbed one of their knives into a foam block repeatedly for 60s. DNA was recovered by mini-taping from triplicate sets of knife handles from four pairings of volunteers after regular use, and at one hour, one day and one week after the handshaking and stabbing events. Total amounts of DNA recovered from the knives, regularly used by a single person, varied among individuals; one volunteer consistently deposited significantly greater amounts than the others, whilst another volunteer did not always leave complete profiles. DNA attributed to the regular user persisted for at least a week, declining with increasing time between DNA deposition and recovery. Non-donor DNA was co-deposited at <5% of the profiles recovered, except for one volunteer, who consistently left DNA from their romantic partner on their knives at ∼25% and ∼11% of the profiles before and after the handshaking and stabbing events, respectively. In three pairings of volunteers, after the handshaking and stabbing events, alleles that could be attributed to the respective handshakers' profiles were detected as partial minor profiles, equating to ∼10% of the profiles recovered. For the fourth pairing of volunteers, only complete single-source DNA profiles matching the regular user's profile were recovered. However, it is important to note that, when indirectly-transferred handshaker DNA was detected, it declined with increasing time between DNA deposition and recovery. These data provide an initial insight into the detection and persistence of directly- and indirectly-transferred DNA that extend the data already available on forensic DNA transfer. The results herein suggest that the sooner an item is sampled after an offence has occurred, the greater the chance of recovering indirectly-transferred DNA, which has implications for forensic reconstructions.

Understanding the factors that may impact the transfer, persistence, prevalence and recovery of DNA (DNA-TPPR), and the availability of data to assign probabilities to DNA quantities and profile types being obtained given particular scenarios and circumstances, is paramount when performing, and giving guidance on, evaluations of DNA findings given activity level propositions (activity level evaluations). In late 2018 and early 2019, three major reviews were published on aspects of DNA-TPPR, with each advocating the need for further research and other actions to support the conduct of DNA-related activity level evaluations. Here, we look at how challenges are being met, primarily by providing a synopsis of DNA-TPPR-related articles published since the conduct of these reviews and briefly exploring some of the actions taken by industry stakeholders towards addressing identified gaps. Much has been carried out in recent years, and efforts continue, to meet the challenges to continually improve the capacity of forensic experts to provide the guidance sought by the judiciary with respect to the transfer of DNA.