Carbon Vacancies Steer the Activity in Dual Ni Carbon Nitride Photocatalysis.

The manipulation of carbon nitride (CN) structures is one main avenue to enhance the activity of CN-based photocatalysts. Increasing the efficiency of photocatalytic heterogeneous materials is a critical step toward the realistic implementation of sustainable schemes for organic synthesis. However, limited knowledge of the structure/activity relationship in relation to subtle structural variations prevents a fully rational design of new photocatalytic materials, limiting practical applications. Here, the CN structure is engineered by means of a microwave treatment, and the structure of the material is shaped around its suitable functionality for Ni dual photocatalysis, with a resulting boosting of the reaction efficiency toward many CX (X = N, S, O) couplings. The combination of advanced characterization techniques and first-principle simulations reveals that this enhanced reactivity is due to the formation of carbon vacancies that evolve into triazole and imine N species able to suitably bind Ni complexes and harness highly efficient dual catalysis. The cost-effective microwave treatment proposed here appears as a versatile and sustainable approach to the design of CN-based photocatalysts for a wide range of industrially relevant organic synthetic reactions.

Humin Formation on SBA-15-pr-SO3H Catalysts during the Alcoholysis of Furfuryl Alcohol to Ethyl Levulinate: Effect of Pore Size on Catalyst Stability, Transport, and Adsorption.

Herein, the alcoholysis of furfuryl alcohol in a series of SBA-15-pr-SO3H catalysts with different pore sizes is reported. Elemental analysis and NMR relaxation/diffusion methods show that changes in pore size have a significant effect on catalyst activity and durability. In particular, the decrease in catalyst activity after catalyst reuse is mainly due to carbonaceous deposition, whereas leaching of sulfonic acid groups is not significant. This effect is more pronounced in the largest-pore-size catalyst C3, which rapidly deactivates after one reaction cycle, whereas catalysts with a relatively medium and small average pore size (named, respectively, C2 and C1) deactivate after two reaction cycles and to a lesser extent. CHNS elemental analysis showed that C1 and C3 experience a similar amount of carbonaceous deposition, suggesting that the increased reusability of the small-pore-size catalyst can be attributed to the presence of SO3H groups mostly present on the external surface, as corroborated by results on pore clogging obtained by NMR relaxation measurements. The increased reusability of the C2 catalyst is attributed to a lower amount of humin being formed and, at the same time, reduced pore clogging, which helps to maintain accessible the internal pore space.

Factors Predicting Outcomes of Supine Percutaneous Nephrolithotomy: Large Single-Centre Experience.

Objective: Percutaneous nephrolithotomy (PCNL) is the treatment of choice for large renal calculi. The prone position has been considered the preferred position to obtain renal access. However, the supine position has recently gained popularity, which confers several potential advantages. The current study analyses the prognostic factors for successful supine PCNL procedures in a larger tertiary centre. Subjects: Prospective data were collected from all patients undergoing PCNL in the Galdako modified Valdivia position at our institution between February-2007 and September-2020. Surgical outcomes variables collected included: the rate of Endoscopic-combined intra-renal surgery (ECIRS), operative times, surgical effectiveness (no residuals <2 mm stone fragments) and complications. Results: A total of 592 patients underwent PCNL with a median age of 56 years (IQR: 42−67). The median stone size was 17 mm (IQR: 13−23). Of those, 79% of patients had an effective procedure. Stone size (p < 0.001), location (p < 0.001) and Guys-Stone Score (GSS) (p < 0.001) were associated with effectiveness. A Percutaneous nephrostomy tube was sited at the completion of the procedure in 97.3% of patients and a simultaneous double-J stent in 45.3%. Stent insertion was associated with larger stones (p < 0.001), the performance of ECIRS (p < 0.001) and higher GSS (p < 0.001). The overall complication rate was 21.7%. The main type of complication was an infection in 26.2 of the cases followed by the need for repeated nephrostogram in 12.7%. Conclusions: We demonstrate that PCNL in a high-volume centre is safe and efficacious in the Galdalko modified Valdivia position. Patients with smaller stones in the renal pelvis and a low GSS have the highest chance of a successful procedure.

Primary Definitive Treatment versus Ureteric Stenting in the Management of Acute Ureteric Colic: A Cost-Effectiveness Analysis.

Objectives: To analyze the differences in cost-effectiveness between primary ureteroscopy and ureteric stenting in patients with ureteric calculi in the emergency setting. Patients and Methods: Patients requiring emergency intervention for a ureteric calculus at a tertiary centre were analysed between January and December 2019. The total secondary care cost included the cost of the procedure, inpatient hospital bed days, emergency department (A&E) reattendances, ancillary procedures and any secondary definitive procedure. Results: A total of 244 patients were included. Patients underwent ureteric stenting (62.3%) or primary treatment (37.7%), including primary ureteroscopy (URS) (34%) and shock wave lithotripsy (SWL) (3.6%). The total secondary care cost was more significant in the ureteric stenting group (GBP 4485.42 vs. GBP 3536.83; p = 0.65), though not statistically significant. While mean procedural costs for primary treatment were significantly higher (GBP 2605.27 vs. GBP 1729.00; p < 0.001), costs in addition to the procedure itself were significantly lower (GBP 931.57 vs. GBP 2742.35; p < 0.001) for primary treatment compared to ureteric stenting. Those undergoing ureteric stenting had a significantly higher A&E reattendance rate compared with primary treatment (25.7% vs. 10.9%, p = 0.02) and a significantly greater cost per patient related to revisits to A&E (GBP 61.05 vs. GBP 20.87; p < 0.001). Conclusion: Primary definitive treatment for patients with acute ureteric colic, although associated with higher procedural costs than ureteric stenting, infers a significant reduction in additional expenses, notably related to fewer A&E attendances. This is particularly relevant in the COVID-19 era, where it is crucial to avoid unnecessary attendances to A&E and reduce the backlog of delayed definitive procedures. Primary treatment should be considered concordance with clinical judgement and factors such as patient preference, equipment availability and operator experience.

Emergency Primary Ureteroscopy for Acute Ureteric Colic-From Guidelines to Practice.

OBJECTIVE: To review the factors that may influence the ability to achieve the present guidelines' recommendations in a well-resourced tertiary centre. According to current National Institute for Health and Care Excellence (NICE) guidelines, definitive treatment (primary ureteroscopy (URS) or shock wave lithotripsy (ESWL)) should be offered to patients with symptomatic renal colic that are unlikely to pass the stone within 48 h of diagnosis. METHODS: Retrospective review of all patients presenting to the emergency department between January and December 2019 with a ureteric or renal stone diagnosis. The rate of emergency intervention, risk factors for intervention and outcomes were compared between patients who were treated by primary definitive surgery vs. primary symptom relief by urethral stenting alone. RESULTS: A total of 244 patients required surgical management for symptomatic ureteric colic without symptoms of urinary infection. Of those, 92 patients (37.7%) underwent definitive treatment by either primary URS (82 patients) or ESWL (9 patients). The mean time for the procedure was 25.5 h (range: 1-118). Patients who underwent primary definitive treatment were likelier to have smaller and distally located stones than the primary stenting group. Primary ureteroscopy was more likely to be performed in a supervised setting than emergency stenting. CONCLUSIONS: Although definitive treatment carries high success rates, in a high-volume tertiary referral centre, it may not be feasible to offer it to all patients, with emergency stenting providing a safe and quick interim measure. Factors determining the ability to provide definitive treatment are stone location, stone size and resident supervision in theatre.

Single-Center Experience with Three Metallic Ureteral Stents (Allium® URS, Memokath™-051, and Resonance®) for Chronic Ureteral Obstruction.

Purpose: Patients with chronic ureteral obstruction (CUO) are traditionally managed with polymer stents/nephrostomy. However, these are prone to failure and require regular exchange. This study evaluates the efficacy of Allium® URS, Memokath™-051, and Resonance® metallic ureteral stents in managing patients with CUO. Methods: Following institutional approval (Imperial College Healthcare NHS Trust, UK, Registration Number SPS_045), operating theater records were reviewed to identify patients with CUO managed with Allium® URS, Memokath™-051, or Resonance® metallic ureteral stents (September 2015/July 2020). Baseline patient variables (age, gender, underlying etiology, and American Society of Anesthesiologists score) and stricture characteristics (length, level, and continuity) were extracted. Intraoperative and postoperative clinical and radiologic assessments at 6 weeks, 3 months, and then every 6 months, as well as any emergency attendances, were reviewed. The primary outcome was duration of functional stent survival. Secondary outcomes included intraoperative placement success and, as an estimate of renal function, mean serum creatinine over time. Results: One hundred twenty-nine stent insertion episodes (SIEs) (Allium® URS: 23; Memokath™-051: 48; Resonance®: 58) occurred in 76 patients (Allium® URS: 16; Memokath™-051: 31; Resonance®: 29). Kaplan-Meier estimates demonstrated that Resonance® provided superior functional stent survival. Overall median actual functional stent follow-up was 11.4 months for Allium® URS, 5.5 months for Memokath™-051, and 11.7 months for Resonance®. 47.8% of Allium® URS (11/23), 64.6% of Memokath™-051 (31/48), and 19% of Resonance® SIEs (11/58) failed. No Resonance® SIEs for benign indication ended in failure. Intraoperative placement success was high (Allium® URS: 95.7%; Memokath™-051 and Resonance®: both 100%). In the first year following SIE, creatinine ranged from +21.3% to +46.7% for Allium® URS, -7.8% to +8.9% for Memokath™-051, and -9.4% to +27.3% for Resonance®. Conclusions: Allium® URS, Memokath™-051, and Resonance® metallic ureteral stents are all viable management options of CUO. In this cohort, Resonance® provided superior functional stent survival. Prospective large-scale comparisons with long-term follow-up are needed to help inform stent choice dependent on individual patient and stricture characteristics.

Orchidopexy for Testicular Torsion: A Systematic Review of Surgical Technique.

CONTEXT: Acute testicular torsion is a common urological emergency. Accepted practice is surgical exploration, detorsion, and orchidopexy for a salvageable testis. OBJECTIVE: To critically evaluate the methods of orchidopexy and their outcomes with a view to determining the optimal surgical technique. EVIDENCE ACQUISITION: This review protocol was published via PROSPERO [CRD42016043165] and conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA). EMBASE, MEDLINE, and CENTRAL databases were searched using the following terms: "orchidopexy", "fixation", "exploration", "torsion", "scrotum", and variants. Article screening was performed by two reviewers independently. The primary outcome was retorsion rate of the ipsilateral testis following orchidopexy. Secondary outcomes included testicular atrophy and fertility. EVIDENCE SYNTHESIS: To our knowledge, this is the first systematic review on this topic. The search yielded 2257 abstracts. Five studies (n = 138 patients) were included. All five techniques differed in incision and/or type of suture and/or point(s) of fixation. Postoperative complications were reported in one study, and included scrotal abscess in 9.1% and stitch abscess in 4.5%. The contralateral testis was fixed in 57.6% of cases. Three studies reported follow-up duration (range 6-31 wk). No study reported any episodes of ipsilateral retorsion. In the studies reporting ipsilateral atrophy rate, this ranged from 9.1% to 47.5%. Fertility outcomes and patient-reported outcome measures were not reported in any studies. CONCLUSIONS: There is limited evidence in favour of any one surgical technique for acute testicular torsion. During the consent process for scrotal exploration, uncertainties in long-term harms should be discussed. This review highlights the need for an interim consensus on surgical approach until robust studies examining the effects of an operative approach on clinical and fertility outcomes are available. PATIENT SUMMARY: Twisting of blood supply to the testis, termed testicular torsion, is a urological emergency. Testicular torsion is treated using an operation to untwist the cord that contains the blood vessels. If the testis is still salvageable, surgery can be performed to prevent further torsion. The method that is used to prevent further torsion varies. We reviewed the literature to assess the outcomes of using various surgical techniques to fix the twisting of the testis. Our review shows that there is limited evidence in favour of any one technique.

The Fate of Ureteral Memokath Stent(s) in a High-Volume Referral Center: An Independent Long-Term Outcomes Review.

Purpose: To independently assess upper urinary tract Memokath (MMK-051) stent outcomes in a national tertiary referral center. Materials and Methods: Two researchers, completely independent to the treating team, reviewed electronic MMK-051 stent(s) patient management records. Outcomes included time to first complication, complication(s)-severity, MMK-051 stent lifespan and change incidence, salvage therapy, further surgical intervention, and mortality. Results and Limitations: One hundred patients received 162 MMK-051 stent(s) (59% with malignant and 63% with distal ureteral obstruction [UO]) with only three lost to follow-up (FU). At 5-year mean FU, only 25 patients had complication-free original MMK-051 stents (14 alive, 11 dead). Of the remaining 75 patients, 22 had other stents, 12 had major surgery (e.g., nephrectomy), 3 became dialysis dependent, and 14 stabilized without ureteral stenting after original MMK-051 removal. Malignant obstruction patients had greater original MMK-051 stent longevity (p < 0.02), but also 20 of the 21 deaths (95%). The 72% mean 5-year stent complication rate included migration (46%), blockage (34%), nonfunctioning kidney (8%), urosepsis needing intravenous antibiotics (8%), and others (6%), including one postoperative death, one ureteral injury, and two with intractable pain. Median time to first complication was 12.5 months. Conclusions: MMK-051 stents had optimal utility in managing malignant UO and in those unfit for corrective surgery. Longer independently assessed mean 5-year outcomes review revealed much higher complication rates (72%) than previously reported. Future international metallic ureteral stent guidelines should encourage clinicians to adopt patient-centered multidisciplinary assessment and selection, with counseling plus goal-setting, and harmonized long-term protocol-based reporting, for optimized future patient safety and outcomes.

Light-driven, heterogeneous organocatalysts for C-C bond formation toward valuable perfluoroalkylated intermediates.

The favorable exploitation of carbon nitride (CN) materials in photocatalysis for organic synthesis requires the appropriate fine-tuning of the CN structure. Here, we present a deep investigation of the structure/activity relationship of CN in the photocatalytic perfluoroalkylation of organic compounds. Four types of CN bearing subtle structural differences were studied via conventional characterization techniques and innovative nuclear magnetic resonance (NMR) experiments, correlating the different structures with the fundamental mechanistic nexus and especially highlighting the importance of the halogen bond strength between the reagent and the catalyst surface. The optimum catalyst exhibited an excellent performance, with a very wide reaction scope, and could prominently trigger the model reaction using natural sunlight. The work lays a platform for establishing a new approach in the development of heterogeneous photocatalysts for organic synthesis related to medical, agricultural, and material chemistry.

Solvent Effects in the Homogeneous Catalytic Reduction of Propionaldehyde with Aluminium Isopropoxide Catalyst: New Insights from PFG NMR and NMR Relaxation Studies.

Solvent effects in homogeneous catalysis are known to affect catalytic activity. Whilst these effects are often described using qualitative features, such as Kamlet-Taft parameters, experimental tools able to quantify and reveal in more depth such effects have remained unexplored. In this work, PFG NMR diffusion and T1 relaxation measurements have been carried out to probe solvent effects in the homogeneous catalytic reduction of propionaldehyde to 1-propanol in the presence of aluminium isopropoxide catalyst. Using data on diffusion coefficients it was possible to estimate trends in aggregation of different solvents. The results show that solvents with a high hydrogen-bond accepting ability, such as ethers, tend to form larger aggregates, which slow down the molecular dynamics of aldehyde molecules, as also suggested by T1 measurements, and preventing their access to the catalytic sites, which results in the observed decrease of catalytic activity. Conversely, weakly interacting solvents, such as alkanes, do not lead to the formation of such aggregates, hence allowing easy access of the aldehyde molecules to the catalytic sites, resulting in higher catalytic activity. The work reported here is a clear example on how combining traditional catalyst screening in homogeneous catalysis with NMR diffusion and relaxation time measurements can lead to new physico-chemical insights into such systems by providing data able to quantify aggregation phenomena and molecular dynamics.

Creation of Al-Enriched Mesoporous ZSM-5 Nanoboxes with High Catalytic Activity: Converting Tetrahedral Extra-Framework Al into Framework Sites by Post Treatment.

ZSM-5 zeolite nanoboxes with accessible meso-micro-pore architecture and strong acid sites are important in relevant heterogeneous catalysis suffering from mass transfer limitations and weak acidities. Rational design of parent zeolites with concentrated and non-protective coordination of Al species can facilitate post-synthetic treatment to produce mesoporous ZSM-5 nanoboxes. In this work, a simple and effective method was developed to convert parent MFI zeolites with tetrahedral extra-framework Al into Al-enriched mesoporous ZSM-5 nanoboxes with low silicon-to-aluminium ratios of ≈16. The parent MFI zeolite was prepared by rapid ageing of the zeolite sol gel synthesis mixture. The accessibility to the meso-micro-porous intra-crystalline network was probed systematically by comparative pulsed field gradient nuclear magnetic resonance diffusion measurements, which, together with the strong acidity of nanoboxes, provided superb catalytic activity and longevity in hydrocarbon cracking for propylene production.

RNF187 is Downregulated Following NF-κB Inhibition in Late Erythroblasts.

Beta (ß)-thalassaemic erythroblasts grown in vitro have reduced nuclear factor kappa B (NF-κB) pathway gene expression. By inhibiting this pathway in erythroblasts from normal individuals, important downstream genes affected by this inhibition can be identified. Bay 11-7082 is a potent inhibitor of the NF-κB pathway, it acts irreversibly, inhibiting NF-κB activation by blocking tumor necrosis factor alpha (TNF-α)-induced phosphorylation of the inhibitory IκB subunit thereby preventing NF-κB activation. In this study, hematopoietic stem cells were isolated from the peripheral blood of 6 healthy individuals and were then cultured for 14 days in conditions which promote erythroid differentiation. Following erythroid lineage enrichment, these cells were stimulated with TNFα or inhibited with Bay 11-7082. Subsequent RNA isolation and gene expression analyses were performed using pooled cDNA with custom PCR arrays. Genes of interest were examined individually on non-pooled samples. Our data identified RNF187, a RING finger domain gene as being downregulated in response to NF-κB inhibition.

The Effect of Nonsense Mediated Decay on Transcriptional Activity Within the Novel ß-Thalassemia Mutation HBB: c.129delT.

Premature termination codons (PTCs) are caused by mutations in the coding sequences of functional genes resulting in an incorrect assignment of a stop codon. Abnormal and truncated proteins are prevented from being translated due to the rapid degradation of mRNA carrying these mutations by an RNA surveillance mechanism referred to as nonsense mediated decay (NMD). Recently, a novel mutation in a patient from Thailand with the clinical diagnosis of Hb E (HBB: c.79G > A)/ß(0)-thalassemia (Hb E/ß(0)-thal) and whose molecular analysis demonstrated a novel mutation in the ß-globin gene, HBB: c.129delT, was reported. The result of this deletion is a frameshift (FSC) resulting in a PTC at codon 60. We have analyzed the impact of this mutation on transcription and translation of the affected ß-globin gene using an in vitro model. The quantitative real-time polymerase chain reaction (qReTi-PCR) analysis revealed that this nucleotide mutation resulted in marked mRNA degradation, which we attributed to the NMD mechanism and as such, the expected deleterious truncated HBB was not generated. This result highlights a valuable application of our in vitro gene expression model that can be used to predict possible molecular pathology for any given nucleotide mutations.

Differential gene expression analysis in early and late erythroid progenitor cells in ß-thalassaemia.

ß- thalassaemia is a disorder of globin gene synthesis resulting in reduced or absent production of the ß-globin chain in red blood cells. In this study, haematopoietic stem cells were isolated from the peripheral blood of six transfusion dependent ß-thalassaemia patients and six healthy controls. Following 7 and 14 d in culture, early- and late- erythroblasts were isolated and purified. No morphological difference in maturation was observed following 7 d in culture, while a delayed maturation was observed in the patient group after 14 d. Following RNA isolation and linear amplification, gene expression analyses were performed using microarray technology. The generated data were analysed by two methods: the BRB-ArrayTools platform and the Bioconductor platform using bead level data. Following 7 d culture, there was no difference in gene expression between the control and patient groups. Following 14 d culture, 384 differentially expressed genes were identified by either analysis. A subset of 90 genes was selected and the results were confirmed by Quantitative-Real-Time-polymerase chain reaction. Pathways shown to be significantly altered in the patient group include apoptosis, MAPKinase and the nuclear factor-κB pathway.

Safety of supervised trainee-performed laparoscopic surgery for inflammatory bowel disease.

PURPOSE: There is a significant risk of surgical resection during the lifetime of an inflammatory bowel disease (IBD) patient: laparoscopic surgery has been increasingly applied to the management of IBD with short and long-term advantages. The aim of this study is to demonstrate that laparoscopic surgery for IBD, performed by a surgical trainee under the supervision of an experienced trainer, is feasible and safe. METHODS: All surgical procedures were sub-divided in six critical steps in order to define the procedure as supervised trainee performed (STP) when the trainer was present unscrubbed in the theatre or assisting and trainer performed (TNER) when the trainer performed two or more critical steps of the procedure. Included were all patients undergoing laparoscopic resection for IBD between January 2009 and December 2013. Thirty-day mortality and morbidity were the primary outcomes. Reoperations and rehospitalizations within 30 days of discharge were recorded prospectively and were the secondary outcomes together with conversion rate and length of hospital stay. RESULTS: One hundred fifty-one patients were included: 77 (50.99%) STP and 74 (49.01%) TNER. No deaths occurred, and 30-day morbidity was 27.15% with no differences between the groups. Operating time was longer in the STP (166.6 ± 53.31 vs 130.4 ± 49.15). Five patients (2 vs 3) required reoperation (3.31%), while 13 patients (8.6%) required readmission. CONCLUSIONS: Laparoscopic surgery for IBD performed by a supervised trainee is safe compared to trainers performed procedures despite a longer operating time. Randomized clinical trials are needed to confirm these preliminary results and to investigate long-term outcomes.

Differential expression of the Bcl-2 and Bax isoforms in CD19 positive B-lymphocytes isolated from patients diagnosed with chronic lymphocytic leukaemia.

AIM: To examine the relative gene expression levels of the anti-apoptotic Bcl-2α and ß isoforms and the pro-apoptotic Baxα and ß isoforms in patients with chronic lymphocytic leukaemia (CLL) and healthy controls (HC). METHODS: Peripheral blood was obtained from 36 patients diagnosed with CLL and 10 HC. CD19 B-lymphocytes were isolated using an antibody coupled magnetic bead isolation system; from these cells the total RNA was isolated and purified. The relative levels of gene expression were examined by quantitative real-time polymerase chain reaction (qReTi-PCR) using primers specific for each isoform. RESULTS: Bcl-2α and Baxα are expressed at higher levels than their ß-isoforms in CLL and HC. Bcl-2α, Bcl-2ß and Baxß expression is increased in CLL while Bax-α is expressed at similar levels to HC. The Bcl-2α/Bcl-2ß ratio is similar in CLL and HC. The Bcl-2α/Baxα ratio is increased in CLL when compared with HC. CONCLUSION: Bcl-2α and Baxα appear to be the dominant anti- and pro-apoptotic isoforms in CLL. The Bcl-2α/Baxα ratio is increased in CLL while the Bcl-2α/Bcl-2ß ratio is similar to HC.

Identification and characterization of two novel and differentially expressed isoforms of human α2- and α1-globin genes.

In most references, the transcription initiation site for the α2- and α1-globin genes has been described to lie 37 bp upstream of the translation initiation codon, however, a review of data repositories such as GenBank and Ensembl showed a report of the α2-globin transcription initiation site occurring at position -66 relative to the initiation codon. To confirm the occurrence of these isoforms for both the α2- and α1-globin genes and to document their expression levels, we initiated our current investigation. Total RNA from the peripheral blood of 15 healthy volunteers was analyzed using both semi-quantitative-polymerase chain reaction (PCR) and real-time (ReTi-PCR) protocols developed in our laboratory, with primers designed to enable distinction between the α2- and α1-globin transcripts.We observed two distinct PCR products for each of the globin genes. Subsequent DNA sequencing of 11 individual PCR products revealed that the α2- and α1-globin transcripts are present in both a long and a short isoform, initiating at positions -66 and -37, respectively. The shorter (-37) isoform is expressed approximately 10,000-100,000 times more strongly than the longer isoform, demonstrating differential expression within the healthy population. This study, for the first time, confirms the presence of two isoforms for both the α2- and α1-globin genes with varying transcription levels in healthy individuals. The short isoform is expressed at significantly higher levels than the longer isoform for both α2 and α1 genes. Therefore, based on our observations, we propose that despite the contribution of the long isoforms to the total α-globin RNA pool, the short isoforms are the main physiological transcripts.

Direct comparison of post-28-cycle PCR purification and modified capillary electrophoresis methods with the 34-cycle "low copy number" (LCN) method for analysis of trace forensic DNA samples.

The investigation of samples with low amounts of template DNA remains at the forefront of forensic DNA research and technology as it becomes increasingly important to gain DNA profile information from exceedingly trace levels of DNA. Previous studies have demonstrated that it is possible to obtain short tandem repeat (STR) profiles from <100pg of template DNA by increasing the number of amplification cycles from 28 to 34, a modification often referred to as "low copy number" or LCN analysis. In this study, we have optimised post-PCR purification techniques applied after only 28 cycles of PCR, as well as using modified capillary electrophoresis injection conditions and have investigated the progressive application of these enhanced approaches. This paper reviews the characteristics of the profiles obtained by these methods compared with those obtained on the same samples after 34-cycle PCR. We observed comparable sensitivity to 34-cycle PCR in terms of the number of profiles with evidence of DNA and the number of allelic peaks per profile and we noted improved peak height and area magnitude with some sample types. Certain parameters reported to be adversely affected in 34-cycle LCN investigations, such as non-donor allele peaks and increased stutter peak ratio, were reduced by this approach. There are a number of advantages for trace samples in progressing from the standard 28-cycle process to the post-PCR processing method as compared to 34-cycle PCR method, including reduced sample consumption, reduced number of PCR amplifications required, and a staged approach to sample processing and profile interpretation.